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Martínez-Montiel N, Vite-Arciniega JDJ, Rosas-Murrieta NH, Martínez-Contreras RD. Repurposing alternative splicing events as potential targets for the design of diagnostic and therapeutic tools in PCa. Front Oncol 2025; 15:1520985. [PMID: 40190563 PMCID: PMC11968427 DOI: 10.3389/fonc.2025.1520985] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/01/2024] [Accepted: 02/04/2025] [Indexed: 04/09/2025] Open
Abstract
Alternative splicing is a key mechanism responsible for protein diversity in eukaryotes. Even when the relevance of this process was initially overlooked, it is now clear that splicing decisions have a strong impact on the physiology of organisms. Moreover, aberrant splicing products have been clearly related to different diseases, including cancer. Deregulation of splicing factors or mutations at the immature mRNA level could be responsible of generating these aberrant products that are involved in cell biology processes, including migration, angiogenesis, differentiation, cell cycle, DNA repair and so on. For this reason, alternative splicing is now considered a hallmark of cancer. Prostate cancer is one of the most frequently diagnosed types of cancer and some of the leading global cause of cancer death men. Prostate cancer shows an important incidence in the developing world, while the mortality rate is growing because of limited medical infrastructure and awareness. Here, we present some of the key alternative splicing events related to prostate cancer and even when the exact role of these isoforms in the development of the disease has not been fully understood, we believe that the correction of these aberrant splicing events represents an attractive target for the design of innovative diagnostic and therapeutic tools.
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Affiliation(s)
- Nancy Martínez-Montiel
- Laboratorio de Ecología Molecular Microbiana, Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Puebla, Mexico
| | - José de Jesús Vite-Arciniega
- Laboratorio de Ecología Molecular Microbiana, Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Puebla, Mexico
| | - Nora Hilda Rosas-Murrieta
- Laboratorio de Bioquímica y Biología Molecular, Centro de Química, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Puebla, Mexico
| | - Rebeca D. Martínez-Contreras
- Laboratorio de Ecología Molecular Microbiana, Centro de Investigaciones en Ciencias Microbiológicas, Instituto de Ciencias, Benemérita Universidad Autónoma de Puebla, Puebla, Mexico
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Peyda P, Lin CH, Onwuzurike K, Black DL. The Rbfox1/LASR complex controls alternative pre-mRNA splicing by recognition of multipart RNA regulatory modules. Genes Dev 2025; 39:364-383. [PMID: 39880658 PMCID: PMC11874969 DOI: 10.1101/gad.352105.124] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2024] [Accepted: 01/06/2025] [Indexed: 01/31/2025]
Abstract
The Rbfox proteins regulate alternative pre-mRNA splicing by binding to the RNA element GCAUG. In the nucleus, most of Rbfox is bound to the large assembly of splicing regulators (LASR), a complex of RNA-binding proteins that recognize additional RNA motifs. However, it remains unclear how the different subunits of the Rbfox/LASR complex act together to bind RNA and regulate splicing. We used a nuclease protection assay to map the transcriptome-wide footprints of Rbfox1/LASR on nascent cellular RNA. In addition to GCAUG, Rbfox1/LASR binds RNA motifs for LASR subunits hnRNPs M, H/F, and C and Matrin3. These elements are often arranged in tandem, forming multipart modules of RNA motifs. To distinguish contact sites of Rbfox1 from the LASR subunits, we analyzed a mutant Rbfox1(F125A) that has lost RNA binding but remains associated with LASR. Rbfox1(F125A)/LASR complexes no longer interact with GCAUG but retain binding to RNA elements for LASR. Splicing analyses reveal that in addition to activating exons through adjacent GCAUG elements, Rbfox can also stimulate exons near binding sites for LASR subunits. Minigene experiments demonstrate that these diverse elements produce a combined regulatory effect on a target exon. These findings illuminate how a complex of RNA-binding proteins can decode combinatorial splicing regulatory signals by recognizing groups of tandem RNA elements.
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Affiliation(s)
- Parham Peyda
- Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, USA
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA
- Medical Scientist Training Program, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California 90095, USA
| | - Chia-Ho Lin
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA
| | - Kelechi Onwuzurike
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA
| | - Douglas L Black
- Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, USA;
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, California 90095, USA
- Jonsson Comprehensive Cancer Center, University of California, Los Angeles, Los Angeles, California 90095, USA
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, Los Angeles, California 90095, USA
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Li J, Qiu H, Dong Q, Yu H, Piao C, Li Z, Sun Y, Cui X. Androgen-targeted hsa_circ_0085121 encodes a novel protein and improves the development of prostate cancer through facilitating the activity of PI3K/Akt/mTOR pathway and enhancing AR-V7 alternative splicing. Cell Death Dis 2024; 15:848. [PMID: 39567496 PMCID: PMC11579034 DOI: 10.1038/s41419-024-07246-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2024] [Revised: 11/10/2024] [Accepted: 11/12/2024] [Indexed: 11/22/2024]
Abstract
Prostate cancer (PCa) is the most prevalent type of cancer and the second leading cause of mortality in males, with a marked increase in incidence observed across the globe. In the present study, whole-transcriptome analysis was conducted to identify differentially expressed circular RNAs (DE-circRNAs). The coding abilities of the DE-circRNAs were analyses, and it was found that hsa_circ_0085121 (circRNF19A) not only exhibited overexpression in PCa cells and tumor samples, but also encoded a 490 amino acid polypeptide designated circRNF19A-490aa. The knockdown of circRNF19A was observed to notably inhibit the proliferation, invasion, migration and docetaxel resistance of PCa cells. In contrast, mutation of the IRES significantly impaired the tumor-promoting function of circRNF19A, indicating that circRNF19A-490aa is the primary form that regulates the malignant behaviors of PCa cells. Mechanistically, circRNF19A-490aa was demonstrated to interact with HSP90AA1, thereby enhancing AR activity and facilitating the activation of the Akt/mTOR and PLK1 pathways. Furthermore, circRNF19A-490aa was observed to interact with HNRNPF, facilitating the recruitment of HNRNPF to the splicing site of AR-V7 and enhancing its alternative splicing. Finally, the androgen receptor (AR) was observed to bind to the promoter region of the RNF19A gene, subsequently regulating the expression of circRNF19A and circRNF19A-490aa. These data indicate that circRNF19A plays a pivotal role in AR activation and AR-V7 generation by encoding a novel protein, circRNF19A-490aa, and targeting circRNF19A may prove an effective strategy for impeding the progression of CRPC.
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Affiliation(s)
- Jianfeng Li
- Department of Urology, First Hospital of China Medical University, #155 Nanjing North Road, 110001, Shenyang, China
| | - Hui Qiu
- Department of Gynecology and Obstetrics, Shengjing Hospital of China Medical University, #36 Sanhao Street, 110004, Shenyang, China
| | - Qingzhuo Dong
- Department of Urology, First Hospital of China Medical University, #155 Nanjing North Road, 110001, Shenyang, China
| | - Hongyuan Yu
- Department of Urology, First Hospital of China Medical University, #155 Nanjing North Road, 110001, Shenyang, China
| | - Chiyuan Piao
- Department of Urology, First Hospital of China Medical University, #155 Nanjing North Road, 110001, Shenyang, China
| | - Zhengxiu Li
- Department of Dermatology, First Hospital of China Medical University, #155 Nanjing North Road, 110001, Shenyang, China
| | - Yanbin Sun
- Department of Thoracic Surgery, First Hospital of China Medical University, #155 Nanjing North Road, 110001, Shenyang, China.
| | - Xiaolu Cui
- Department of Urology, First Hospital of China Medical University, #155 Nanjing North Road, 110001, Shenyang, China.
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Netherton JK, Ogle RA, Robinson BR, Molloy M, Krisp C, Velkov T, Casagranda F, Dominado N, Silva Balbin Villaverde AI, Zhang XD, Hime GR, Baker MA. The role of HnrnpF/H as a driver of oligoteratozoospermia. iScience 2024; 27:110198. [PMID: 39092172 PMCID: PMC11292545 DOI: 10.1016/j.isci.2024.110198] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2023] [Revised: 02/20/2024] [Accepted: 06/03/2024] [Indexed: 08/04/2024] Open
Abstract
Male subfertility or infertility is a common condition often characterized by men producing a low number of sperm with poor quality. To gain insight into this condition, we performed a quantitative proteomic analysis of semen samples obtained from infertile and fertile men. At least 6 proteins showed significant differences in regulation of alternatively spliced isoforms. To investigate this link between aberrant alternative splicing and production of poor-quality spermatozoa, we overexpressed the hnrnpH/F-orthologue Glorund (Glo) in Drosophila, which was also found to be abundant in poor quality human sperm. Transgenic animals produced low numbers of morphologically defective spermatozoa and aberrant formation of the "dense body," an organelle akin to the mammalian manchette. Furthermore, fertility trials demonstrated that transgenic flies were either completely infertile or highly subfertile. These findings suggest that dysregulation of hnrnpH/F is likely to result in the production of low-quality semen, leading to subfertility or infertility in men.
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Affiliation(s)
- Jacob K. Netherton
- School of Biomedical Sciences and Pharmacy, Faculty of Medicine and Health, University of Newcastle, Callaghan, NSW 2308, Australia
| | - Rachel A. Ogle
- School of Biomedical Sciences and Pharmacy, Faculty of Medicine and Health, University of Newcastle, Callaghan, NSW 2308, Australia
| | - Benjamin R. Robinson
- School of Biomedical Sciences and Pharmacy, Faculty of Medicine and Health, University of Newcastle, Callaghan, NSW 2308, Australia
| | - Mark Molloy
- Australian Proteome Analysis Facility, Department of Biomolecular Sciences, Macquarie University, NSW 2109 Australia
| | - Christoph Krisp
- Australian Proteome Analysis Facility, Department of Biomolecular Sciences, Macquarie University, NSW 2109 Australia
| | - Tony Velkov
- Biomedicine Discovery Institute, Infection & Immunity Program and Department of Microbiology, Monash University, Clayton, VIC 3168, Australia
| | - Franca Casagranda
- Department of Anatomy and Physiology, University of Melbourne, Parkville, VIC 3010, Australia
| | - Nicole Dominado
- Department of Anatomy and Physiology, University of Melbourne, Parkville, VIC 3010, Australia
| | | | - Xu Dong Zhang
- School of Biomedical Sciences and Pharmacy, Faculty of Medicine and Health, University of Newcastle, Callaghan, NSW 2308, Australia
| | - Gary R. Hime
- Department of Anatomy and Physiology, University of Melbourne, Parkville, VIC 3010, Australia
| | - Mark A. Baker
- School of Biomedical Sciences and Pharmacy, Faculty of Medicine and Health, University of Newcastle, Callaghan, NSW 2308, Australia
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Peyda P, Lin CH, Onwuzurike K, Black DL. The Rbfox1/LASR complex controls alternative pre-mRNA splicing by recognition of multi-part RNA regulatory modules. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.07.12.603345. [PMID: 39071271 PMCID: PMC11275806 DOI: 10.1101/2024.07.12.603345] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/30/2024]
Abstract
The Rbfox proteins regulate alternative pre-mRNA splicing by binding to the RNA element GCAUG. In the nucleus, most of Rbfox is bound to LASR, a complex of RNA-binding proteins that recognize additional RNA motifs. However, it remains unclear how the different subunits of the Rbfox/LASR complex act together to bind RNA and regulate splicing. We used a nuclease-protection assay to map the transcriptome-wide footprints of Rbfox1/LASR on nascent cellular RNA. In addition to GCAUG, Rbfox1/LASR binds RNA containing motifs for LASR subunits hnRNPs M, H/F, C, and Matrin3. These elements are often arranged in tandem, forming multi-part modules of RNA motifs. To distinguish contact sites of Rbfox1 from the LASR subunits, we analyzed a mutant Rbfox1(F125A) that has lost RNA binding but remains associated with LASR. Rbfox1(F125A)/LASR complexes no longer interact with GCAUG but retain binding to RNA elements for LASR. Splicing analyses reveal that in addition to activating exons through adjacent GCAUG elements, Rbfox can also stimulate exons near binding sites for LASR subunits. Mini-gene experiments demonstrate that these diverse elements produce a combined regulatory effect on a target exon. These findings illuminate how a complex of RNA-binding proteins can decode combinatorial splicing regulatory signals by recognizing groups of tandem RNA elements.
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Zhao B, Deng J, Ma M, Li N, Zhou J, Li X, Luan T. Environmentally relevant concentrations of 2,3,7,8-TCDD induced inhibition of multicellular alternative splicing and transcriptional dysregulation. THE SCIENCE OF THE TOTAL ENVIRONMENT 2024; 919:170892. [PMID: 38346650 DOI: 10.1016/j.scitotenv.2024.170892] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/13/2023] [Revised: 02/07/2024] [Accepted: 02/09/2024] [Indexed: 02/17/2024]
Abstract
Alternative splicing (AS), found in approximately 95 % of human genes, significantly amplifies protein diversity and is implicated in disease pathogenesis when dysregulated. However, the precise involvement of AS in the toxic mechanisms induced by TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) remains incompletely elucidated. This study conducted a thorough global AS analysis in six human cell lines following TCDD exposure. Our findings revealed that environmentally relevant concentration (0.1 nM) of TCDD significantly suppressed AS events in all cell types, notably inhibiting diverse splicing events and reducing transcript diversity, potentially attributed to modifications in the splicing patterns of the inhibitory factor family, particularly hnRNP. And we identified 151 genes with substantial AS alterations shared among these cell types, particularly enriched in immune and metabolic pathways. Moreover, TCDD induced cell-specific changes in splicing patterns and transcript levels, with increased sensitivity notably in THP-1 monocyte, potentially linked to aberrant expression of pivotal genes within the spliceosome pathway (DDX5, EFTUD2, PUF60, RBM25, SRSF1, and CRNKL1). This study extends our understanding of disrupted alternative splicing and its relation to the multisystem toxicity of TCDD. It sheds light on how environmental toxins affect post-transcriptional regulatory processes, offering a fresh perspective for toxicology and disease etiology investigations.
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Affiliation(s)
- Bilin Zhao
- Guangdong Provincial Laboratory of Chemistry and Fine Chemical Engineering Jieyang Center, Jieyang 515200, China; Guangdong Provincial Key Laboratory of Water Quality Improvement and Ecological Restoration for Watersheds, School of Ecology, Environment and Resources, Guangdong University of Technology, Guangzhou 510006, China
| | - Jiewei Deng
- Guangdong Provincial Laboratory of Chemistry and Fine Chemical Engineering Jieyang Center, Jieyang 515200, China; School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, China; Smart Medical Innovation Technology Center, Guangdong University of Technology, Guangzhou 510006, China
| | - Mei Ma
- State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
| | - Na Li
- State Key Laboratory of Environmental Aquatic Chemistry, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
| | - Junlin Zhou
- Guangdong Provincial Key Laboratory of Water Quality Improvement and Ecological Restoration for Watersheds, School of Ecology, Environment and Resources, Guangdong University of Technology, Guangzhou 510006, China
| | - Xinyan Li
- Guangdong Provincial Laboratory of Chemistry and Fine Chemical Engineering Jieyang Center, Jieyang 515200, China; School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, China; Smart Medical Innovation Technology Center, Guangdong University of Technology, Guangzhou 510006, China.
| | - Tiangang Luan
- Guangdong Provincial Laboratory of Chemistry and Fine Chemical Engineering Jieyang Center, Jieyang 515200, China; Guangdong Provincial Key Laboratory of Water Quality Improvement and Ecological Restoration for Watersheds, School of Ecology, Environment and Resources, Guangdong University of Technology, Guangzhou 510006, China; Smart Medical Innovation Technology Center, Guangdong University of Technology, Guangzhou 510006, China
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7
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Le Sénéchal R, Keruzoré M, Quillévéré A, Loaëc N, Dinh VT, Reznichenko O, Guixens-Gallardo P, Corcos L, Teulade-Fichou MP, Granzhan A, Blondel M. Alternative splicing of BCL-x is controlled by RBM25 binding to a G-quadruplex in BCL-x pre-mRNA. Nucleic Acids Res 2023; 51:11239-11257. [PMID: 37811881 PMCID: PMC10639069 DOI: 10.1093/nar/gkad772] [Citation(s) in RCA: 9] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2023] [Revised: 08/05/2023] [Accepted: 09/09/2023] [Indexed: 10/10/2023] Open
Abstract
BCL-x is a master regulator of apoptosis whose pre-mRNA is alternatively spliced into either a long (canonical) anti-apoptotic Bcl-xL isoform, or a short (alternative) pro-apoptotic Bcl-xS isoform. The balance between these two antagonistic isoforms is tightly regulated and overexpression of Bcl-xL has been linked to resistance to chemotherapy in several cancers, whereas overexpression of Bcl-xS is associated to some forms of diabetes and cardiac disorders. The splicing factor RBM25 controls alternative splicing of BCL-x: its overexpression favours the production of Bcl-xS, whereas its downregulation has the opposite effect. Here we show that RBM25 directly and specifically binds to GQ-2, an RNA G-quadruplex (rG4) of BCL-x pre-mRNA that forms at the vicinity of the alternative 5' splice site leading to the alternative Bcl-xS isoform. This RBM25/rG4 interaction is crucial for the production of Bcl-xS and depends on the RE (arginine-glutamate-rich) motif of RBM25, thus defining a new type of rG4-interacting domain. PhenDC3, a benchmark G4 ligand, enhances the binding of RBM25 to the GQ-2 rG4 of BCL-x pre-mRNA, thereby promoting the alternative pro-apoptotic Bcl-xS isoform and triggering apoptosis. Furthermore, the screening of a combinatorial library of 90 putative G4 ligands led to the identification of two original compounds, PhenDH8 and PhenDH9, superior to PhenDC3 in promoting the Bcl-xS isoform and apoptosis. Thus, favouring the interaction between RBM25 and the GQ-2 rG4 of BCL-x pre-mRNA represents a relevant intervention point to re-sensitize cancer cells to chemotherapy.
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Affiliation(s)
- Ronan Le Sénéchal
- Univ Brest; Inserm UMR1078; Etablissement Français du Sang (EFS) Bretagne; CHRU Brest, Hôpital Morvan, Laboratoire de Génétique Moléculaire, 22 avenue Camille Desmoulins, F-29200 Brest, France
| | - Marc Keruzoré
- Univ Brest; Inserm UMR1078; Etablissement Français du Sang (EFS) Bretagne; CHRU Brest, Hôpital Morvan, Laboratoire de Génétique Moléculaire, 22 avenue Camille Desmoulins, F-29200 Brest, France
| | - Alicia Quillévéré
- Univ Brest; Inserm UMR1078; Etablissement Français du Sang (EFS) Bretagne; CHRU Brest, Hôpital Morvan, Laboratoire de Génétique Moléculaire, 22 avenue Camille Desmoulins, F-29200 Brest, France
| | - Nadège Loaëc
- Univ Brest; Inserm UMR1078; Etablissement Français du Sang (EFS) Bretagne; CHRU Brest, Hôpital Morvan, Laboratoire de Génétique Moléculaire, 22 avenue Camille Desmoulins, F-29200 Brest, France
| | - Van-Trang Dinh
- Univ Brest; Inserm UMR1078; Etablissement Français du Sang (EFS) Bretagne; CHRU Brest, Hôpital Morvan, Laboratoire de Génétique Moléculaire, 22 avenue Camille Desmoulins, F-29200 Brest, France
| | - Oksana Reznichenko
- Chemistry and Modelling for the Biology of Cancer (CMBC), CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris Saclay, F-91405 Orsay, France
| | - Pedro Guixens-Gallardo
- Chemistry and Modelling for the Biology of Cancer (CMBC), CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris Saclay, F-91405 Orsay, France
| | - Laurent Corcos
- Univ Brest; Inserm UMR1078; Etablissement Français du Sang (EFS) Bretagne; CHRU Brest, Hôpital Morvan, Laboratoire de Génétique Moléculaire, 22 avenue Camille Desmoulins, F-29200 Brest, France
| | - Marie-Paule Teulade-Fichou
- Chemistry and Modelling for the Biology of Cancer (CMBC), CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris Saclay, F-91405 Orsay, France
| | - Anton Granzhan
- Chemistry and Modelling for the Biology of Cancer (CMBC), CNRS UMR9187, Inserm U1196, Institut Curie, Université Paris Saclay, F-91405 Orsay, France
| | - Marc Blondel
- Univ Brest; Inserm UMR1078; Etablissement Français du Sang (EFS) Bretagne; CHRU Brest, Hôpital Morvan, Laboratoire de Génétique Moléculaire, 22 avenue Camille Desmoulins, F-29200 Brest, France
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Farshadyeganeh P, Nazim M, Zhang R, Ohkawara B, Nakajima K, Rahman MA, Nasrin F, Ito M, Takeda JI, Ohe K, Miyasaka Y, Ohno T, Masuda A, Ohno K. Splicing regulation of GFPT1 muscle-specific isoform and its roles in glucose metabolisms and neuromuscular junction. iScience 2023; 26:107746. [PMID: 37744035 PMCID: PMC10514471 DOI: 10.1016/j.isci.2023.107746] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2023] [Revised: 07/29/2023] [Accepted: 08/24/2023] [Indexed: 09/26/2023] Open
Abstract
Glutamine:fructose-6-phosphate transaminase 1 (GFPT1) is the rate-limiting enzyme of the hexosamine biosynthetic pathway (HBP). A 54-bp exon 9 of GFPT1 is specifically included in skeletal and cardiac muscles to generate a long isoform of GFPT1 (GFPT1-L). We showed that SRSF1 and Rbfox1/2 cooperatively enhance, and hnRNP H/F suppresses, the inclusion of human GFPT1 exon 9 by modulating recruitment of U1 snRNP. Knockout (KO) of GFPT1-L in skeletal muscle markedly increased the amounts of GFPT1 and UDP-HexNAc, which subsequently suppressed the glycolytic pathway. Aged KO mice showed impaired insulin-mediated glucose uptake, as well as muscle weakness and fatigue likely due to abnormal formation and maintenance of the neuromuscular junction. Taken together, GFPT1-L is likely to be acquired in evolution in mammalian striated muscles to attenuate the HBP for efficient glycolytic energy production, insulin-mediated glucose uptake, and the formation and maintenance of the neuromuscular junction.
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Affiliation(s)
- Paniz Farshadyeganeh
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Mohammad Nazim
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA
| | - Ruchen Zhang
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Bisei Ohkawara
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Kazuki Nakajima
- Institute for Glyco-core Research (iGCORE), Gifu University, Gifu 501-1193, Japan
| | - Mohammad Alinoor Rahman
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
- Department of Biochemistry and Molecular Biology, Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences (UAMS), Little Rock, AR 72205, USA
| | - Farhana Nasrin
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
- Department of Biochemistry and Molecular Biology, Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences (UAMS), Little Rock, AR 72205, USA
| | - Mikako Ito
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Jun-ichi Takeda
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Kenji Ohe
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
- Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan
| | - Yuki Miyasaka
- Division of Experimental Animals, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Tamio Ohno
- Division of Experimental Animals, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Akio Masuda
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
| | - Kinji Ohno
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
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9
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Brownmiller T, Caplen NJ. The HNRNPF/H RNA binding proteins and disease. WILEY INTERDISCIPLINARY REVIEWS. RNA 2023; 14:e1788. [PMID: 37042074 PMCID: PMC10523889 DOI: 10.1002/wrna.1788] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/31/2022] [Revised: 02/10/2023] [Accepted: 02/13/2023] [Indexed: 04/13/2023]
Abstract
The members of the HNRNPF/H family of heterogeneous nuclear RNA proteins-HNRNPF, HNRNPH1, HNRNPH2, HNRNPH3, and GRSF1, are critical regulators of RNA maturation. Documented functions of these proteins include regulating splicing, particularly alternative splicing, 5' capping and 3' polyadenylation of RNAs, and RNA export. The assignment of these proteins to the HNRNPF/H protein family members relates to differences in the amino acid composition of their RNA recognition motifs, which differ from those of other RNA binding proteins (RBPs). HNRNPF/H proteins typically bind RNA sequences enriched with guanine (G) residues, including sequences that, in the presence of a cation, have the potential to form higher-order G-quadruplex structures. The need to further investigate members of the HNRNPF/H family of RBPs has intensified with the recent descriptions of their involvement in several disease states, including the pediatric tumor Ewing sarcoma and the hematological malignancy mantle cell lymphoma; newly described groups of developmental syndromes; and neuronal-related disorders, including addictive behavior. Here, to foster the study of the HNRNPF/H family of RBPs, we discuss features of the genes encoding these proteins, their structures and functions, and emerging contributions to disease. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Processing > Splicing Regulation/Alternative Splicing RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.
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Affiliation(s)
- Tayvia Brownmiller
- Functional Genetics Section, Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, DHHS, Bethesda, Maryland, USA
| | - Natasha J Caplen
- Functional Genetics Section, Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, DHHS, Bethesda, Maryland, USA
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10
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Chen X, Yang HT, Zhang B, Phillips JW, Cheng D, Rigo F, Witte ON, Xing Y, Black DL. The RNA-binding proteins hnRNP H and F regulate splicing of a MYC-dependent HRAS exon in prostate cancer cells. Proc Natl Acad Sci U S A 2023; 120:e2220190120. [PMID: 37399401 PMCID: PMC10334793 DOI: 10.1073/pnas.2220190120] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2022] [Accepted: 05/31/2023] [Indexed: 07/05/2023] Open
Abstract
The MYC proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by MYC transcriptionally up-regulates the core pre-mRNA splicing machinery and causes misregulation of alternative splicing. However, our understanding of how splicing changes are directed by MYC is limited. We performed a signaling pathway-guided splicing analysis to identify MYC-dependent splicing events. These included an HRAS cassette exon repressed by MYC across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA-binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of HNRNPH gene expression with MYC hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, HNRNPF expression showed a positive correlation with MYC hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal mechanisms for MYC-dependent regulation of splicing and point to possible therapeutic targets in prostate cancers.
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Affiliation(s)
- Xinyuan Chen
- Molecular Biology Interdepartmental Doctoral Program, University of California, Los Angeles, CA90095
| | - Harry Taegyun Yang
- Bioinformatics Interdepartmental Graduate Program, University of California, Los Angeles, CA90095
| | - Beatrice Zhang
- Center for Computational and Genomic Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA19104
| | - John W. Phillips
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA90095
| | - Donghui Cheng
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA90095
| | - Frank Rigo
- Ionis Pharmaceuticals, Inc., 2855 Gazelle Ct., Carlsbad, CA92010
| | - Owen N. Witte
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA90095
- Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA90095
- Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA90095
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA90095
- Molecular Biology Institute, University of California, Los Angeles, CA90095
| | - Yi Xing
- Center for Computational and Genomic Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA19104
- Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA19104
| | - Douglas L. Black
- Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA90095
- Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA90095
- Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA90095
- Molecular Biology Institute, University of California, Los Angeles, CA90095
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11
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Huang H, Li Y, Zhang G, Ruan GX, Zhu Z, Chen W, Zou J, Zhang R, Wang J, Ouyang Y, Xu S, Ou X. The RNA-binding protein hnRNP F is required for the germinal center B cell response. Nat Commun 2023; 14:1731. [PMID: 36997512 PMCID: PMC10063658 DOI: 10.1038/s41467-023-37308-z] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2022] [Accepted: 03/10/2023] [Indexed: 04/01/2023] Open
Abstract
The T cell-dependent (TD) antibody response involves the generation of high affinity, immunoglobulin heavy chain class-switched antibodies that are generated through germinal center (GC) response. This process is controlled by coordinated transcriptional and post-transcriptional gene regulatory mechanisms. RNA-binding proteins (RBPs) have emerged as critical players in post-transcriptional gene regulation. Here we demonstrate that B cell-specific deletion of RBP hnRNP F leads to diminished production of class-switched antibodies with high affinities in response to a TD antigen challenge. B cells deficient in hnRNP F are characterized by defective proliferation and c-Myc upregulation upon antigenic stimulation. Mechanistically, hnRNP F directly binds to the G-tracts of Cd40 pre-mRNA to promote the inclusion of Cd40 exon 6 that encodes its transmembrane domain, thus enabling appropriate CD40 cell surface expression. Furthermore, we find that hnRNP A1 and A2B1 can bind to the same region of Cd40 pre-mRNA but suppress exon 6 inclusion, suggesting that these hnRNPs and hnRNP F might antagonize each-other's effects on Cd40 splicing. In summary, our study uncovers an important posttranscriptional mechanism regulating the GC response.
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Affiliation(s)
- Hengjun Huang
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, 518055, China
| | - Yuxing Li
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, 518055, China
| | - Gaopu Zhang
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, 518055, China
| | - Gui-Xin Ruan
- Medical School, Taizhou University, Taizhou, 318000, China
| | - Zhijian Zhu
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, 518055, China
| | - Wenjing Chen
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, 518055, China
| | - Jia Zou
- Department of Computer Science and Engineering, College of Engineering, Southern University of Science and Technology, Shenzhen, 518055, China
| | - Rui Zhang
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, 518055, China
| | - Jing Wang
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, 518055, China
| | - Yu Ouyang
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, 518055, China
| | - Shengli Xu
- Singapore Immunology Network, Agency for Science, Technology and Research, Singapore, 138648, Singapore.
- Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117599, Singapore.
| | - Xijun Ou
- School of Life Sciences, Southern University of Science and Technology, Shenzhen, 518055, China.
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12
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Abstract
Dysregulated RNA splicing is a molecular feature that characterizes almost all tumour types. Cancer-associated splicing alterations arise from both recurrent mutations and altered expression of trans-acting factors governing splicing catalysis and regulation. Cancer-associated splicing dysregulation can promote tumorigenesis via diverse mechanisms, contributing to increased cell proliferation, decreased apoptosis, enhanced migration and metastatic potential, resistance to chemotherapy and evasion of immune surveillance. Recent studies have identified specific cancer-associated isoforms that play critical roles in cancer cell transformation and growth and demonstrated the therapeutic benefits of correcting or otherwise antagonizing such cancer-associated mRNA isoforms. Clinical-grade small molecules that modulate or inhibit RNA splicing have similarly been developed as promising anticancer therapeutics. Here, we review splicing alterations characteristic of cancer cell transcriptomes, dysregulated splicing's contributions to tumour initiation and progression, and existing and emerging approaches for targeting splicing for cancer therapy. Finally, we discuss the outstanding questions and challenges that must be addressed to translate these findings into the clinic.
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Affiliation(s)
- Robert K Bradley
- Computational Biology Program, Public Health Sciences Division and Basic Sciences Division, Fred Hutchinson Cancer Center, Seattle, WA, USA.
| | - Olga Anczuków
- The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA.
- Department of Genetics and Genome Sciences, UConn Health, Farmington, CT, USA.
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13
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Wu W, Chen A, Lin S, Wang Q, Lian G, Luo L, Xie L. The identification and verification of hub genes associated with pulmonary arterial hypertension using weighted gene co-expression network analysis. BMC Pulm Med 2022; 22:474. [PMID: 36514015 PMCID: PMC9746192 DOI: 10.1186/s12890-022-02275-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2022] [Accepted: 12/05/2022] [Indexed: 12/15/2022] Open
Abstract
BACKGROUND Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance and pulmonary arterial pressure, with complex etiology, difficult treatment and poor prognosis. The objective of this study was to investigate the potential biomarkers for PAH based on bioinformatics analysis. METHODS The GSE117261 datasets were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified by screening PAH patients and controls. Then the DEGs were analyzed using a Weighted Gene Co-expression Network Analysis (WGCNA) and the key modules were determined, and to further explore their potential biological functions via Gene Ontology analysis (GO), Kyoto Encyclopedia of Genes and Genomes Pathway analysis (KEGG), and Gene Set Enrichment Analysis (GSEA). Moreover, Protein-protein interaction (PPI) networks were constructed to identify hub gene candidates in the key modules. Finally, real-time quantitative polymerase chain reaction was supplied to detect the expressions of hub genes in human pulmonary arterial smooth cells treated with cobalt chloride (COCl2) which was used to mimic hypoxia. RESULTS There were 2299 DEGs identified. WGCNA indicated that yellow module was the key one correlated with PAH. GO and KEGG analysis demonstrated that genes in the yellow module were mainly enriched in 'Pathways in cancer'. GSEA revealed that 'HALLMARK_MYC_TARGETS_V1' was remarkably enriched in PAH. Based on the PPI network, vascular endothelial growth factor A, proto-oncogene receptor tyrosine kinase (KIT), PNN interacting serine and arginine rich protein (PNISR) and heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) were identified as the hub genes. Additionally, the PCR indicated that the elevated expressions of PNISR and HNRNPH1 were in line with the bioinformatics analysis. ROC analysis determined that PNISR and HNRNPH1 may be potential biomarkers to provide better diagnosis of PAH. CONCLUSION PNISR and HNRNPH1 were potential biomarkers to diagnosis PAH. In summary, the identified DEGs, modules, pathways, and hub genes provide clues and shed light on the potential molecular mechanisms of PAH.
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Affiliation(s)
- Weibin Wu
- grid.412683.a0000 0004 1758 0400Department of Geriatrics, The First Affiliated Hospital of Fujian Medical University, 20 Chazhong Road, Fuzhou, 350005 Fujian People’s Republic of China ,grid.412683.a0000 0004 1758 0400Fujian Hypertension Research Institute, The First Affiliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China ,grid.412683.a0000 0004 1758 0400Clinical Research Center for Geriatric Hypertension Disease of Fujian Province, The First Affiliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China ,grid.412683.a0000 0004 1758 0400Branch of National Clinical Research Center for Aging and Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian People’s Republic of China ,grid.256112.30000 0004 1797 9307Department of Geriatrics, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou, People’s Republic of China
| | - Ai Chen
- grid.412683.a0000 0004 1758 0400Department of Geriatrics, The First Affiliated Hospital of Fujian Medical University, 20 Chazhong Road, Fuzhou, 350005 Fujian People’s Republic of China ,grid.412683.a0000 0004 1758 0400Fujian Hypertension Research Institute, The First Affiliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China ,grid.412683.a0000 0004 1758 0400Clinical Research Center for Geriatric Hypertension Disease of Fujian Province, The First Affiliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China ,grid.412683.a0000 0004 1758 0400Branch of National Clinical Research Center for Aging and Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian People’s Republic of China ,grid.256112.30000 0004 1797 9307Department of Geriatrics, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou, People’s Republic of China
| | - Siming Lin
- grid.412683.a0000 0004 1758 0400Fujian Hypertension Research Institute, The First Affiliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China
| | - Qiuran Wang
- grid.412683.a0000 0004 1758 0400Department of Geriatrics, The First Affiliated Hospital of Fujian Medical University, 20 Chazhong Road, Fuzhou, 350005 Fujian People’s Republic of China ,grid.412683.a0000 0004 1758 0400Fujian Hypertension Research Institute, The First Affiliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China ,grid.412683.a0000 0004 1758 0400Clinical Research Center for Geriatric Hypertension Disease of Fujian Province, The First Affiliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China ,grid.412683.a0000 0004 1758 0400Branch of National Clinical Research Center for Aging and Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian People’s Republic of China ,grid.256112.30000 0004 1797 9307Department of Geriatrics, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou, People’s Republic of China
| | - Guili Lian
- grid.412683.a0000 0004 1758 0400Department of Geriatrics, The First Affiliated Hospital of Fujian Medical University, 20 Chazhong Road, Fuzhou, 350005 Fujian People’s Republic of China ,grid.412683.a0000 0004 1758 0400Fujian Hypertension Research Institute, The First Affiliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China ,grid.412683.a0000 0004 1758 0400Clinical Research Center for Geriatric Hypertension Disease of Fujian Province, The First Affiliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China ,grid.412683.a0000 0004 1758 0400Branch of National Clinical Research Center for Aging and Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian People’s Republic of China ,grid.256112.30000 0004 1797 9307Department of Geriatrics, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou, People’s Republic of China
| | - Li Luo
- grid.412683.a0000 0004 1758 0400Department of Geriatrics, The First Affiliated Hospital of Fujian Medical University, 20 Chazhong Road, Fuzhou, 350005 Fujian People’s Republic of China ,grid.412683.a0000 0004 1758 0400Fujian Hypertension Research Institute, The First Affiliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China ,grid.412683.a0000 0004 1758 0400Clinical Research Center for Geriatric Hypertension Disease of Fujian Province, The First Affiliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China ,grid.412683.a0000 0004 1758 0400Branch of National Clinical Research Center for Aging and Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian People’s Republic of China ,grid.256112.30000 0004 1797 9307Department of Geriatrics, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou, People’s Republic of China
| | - Liangdi Xie
- grid.412683.a0000 0004 1758 0400Department of Geriatrics, The First Affiliated Hospital of Fujian Medical University, 20 Chazhong Road, Fuzhou, 350005 Fujian People’s Republic of China ,grid.412683.a0000 0004 1758 0400Fujian Hypertension Research Institute, The First Affiliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China ,grid.412683.a0000 0004 1758 0400Clinical Research Center for Geriatric Hypertension Disease of Fujian Province, The First Affiliated Hospital of Fujian Medical University, Fuzhou, People’s Republic of China ,grid.412683.a0000 0004 1758 0400Branch of National Clinical Research Center for Aging and Medicine, The First Affiliated Hospital of Fujian Medical University, Fuzhou, Fujian People’s Republic of China ,grid.256112.30000 0004 1797 9307Department of Geriatrics, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou, People’s Republic of China
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Shkreta L, Delannoy A, Salvetti A, Chabot B. SRSF10: an atypical splicing regulator with critical roles in stress response, organ development, and viral replication. RNA (NEW YORK, N.Y.) 2021; 27:1302-1317. [PMID: 34315816 PMCID: PMC8522700 DOI: 10.1261/rna.078879.121] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 06/13/2023]
Abstract
Serine/arginine splicing factor 10 (SRSF10) is a member of the family of mammalian splicing regulators known as SR proteins. Like several of its SR siblings, the SRSF10 protein is composed of an RNA binding domain (RRM) and of arginine and serine-rich auxiliary domains (RS) that guide interactions with other proteins. The phosphorylation status of SRSF10 is of paramount importance for its activity and is subjected to changes during mitosis, heat-shock, and DNA damage. SRSF10 overexpression has functional consequences in a growing list of cancers. By controlling the alternative splicing of specific transcripts, SRSF10 has also been implicated in glucose, fat, and cholesterol metabolism, in the development of the embryonic heart, and in neurological processes. SRSF10 is also important for the proper expression and processing of HIV-1 and other viral transcripts. We discuss how SRSF10 could become a potentially appealing therapeutic target to combat cancer and viral infections.
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Affiliation(s)
- Lulzim Shkreta
- RNA group, Department of Microbiology and Infectious Diseases, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada J1E 4K8
| | - Aurélie Delannoy
- RNA group, Department of Microbiology and Infectious Diseases, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada J1E 4K8
| | - Anna Salvetti
- INSERM, U1111, Centre International de Recherche en Infectiologie de Lyon (CIRI), CNRS UMR 5308, Lyon, France
| | - Benoit Chabot
- RNA group, Department of Microbiology and Infectious Diseases, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada J1E 4K8
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15
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Liu M, Yang L, Liu X, Nie Z, Zhang X, Lu Y, Pan Y, Wang X, Luo J. HNRNPH1 Is a Novel Regulator Of Cellular Proliferation and Disease Progression in Chronic Myeloid Leukemia. Front Oncol 2021; 11:682859. [PMID: 34295818 PMCID: PMC8290130 DOI: 10.3389/fonc.2021.682859] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2021] [Accepted: 06/17/2021] [Indexed: 12/29/2022] Open
Abstract
RNA binding proteins act as essential modulators in cancers by regulating biological cellular processes. Heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1), as a key member of the heterogeneous nuclear ribonucleoproteins family, is frequently upregulated in multiple cancer cells and involved in tumorigenesis. However, the function of HNRNPH1 in chronic myeloid leukemia (CML) remains unclear. In the present study, we revealed that HNRNPH1 expression level was upregulated in CML patients and cell lines. Moreover, the higher level of HNRNPH1 was correlated with disease progression of CML. In vivo and in vitro experiments showed that knockdown of HNRNPH1 inhibited cell proliferation and promoted cell apoptosis in CML cells. Importantly, knockdown of HNRNPH1 in CML cells enhanced sensitivity to imatinib. Mechanically, HNRNPH1 could bind to the mRNA of PTPN6 and negatively regulated its expression. PTPN6 mediated the regulation between HNRNPH1 and PI3K/AKT activation. Furthermore, the HNRNPH1–PTPN6–PI3K/AKT axis played a critical role in CML tumorigenesis and development. The present study first investigated the deregulated HNRNPH1–PTPN6–PI3K/AKT axis moderated cell growth and apoptosis in CML cells, whereby targeting this pathway may be a therapeutic CML treatment.
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Affiliation(s)
- Menghan Liu
- Department of Hematology, The Second Hospital of Hebei Medical University, Key Laboratory of Hematology, Shijiazhuang, China
| | - Lin Yang
- Department of Hematology, The Second Hospital of Hebei Medical University, Key Laboratory of Hematology, Shijiazhuang, China
| | - Xiaojun Liu
- Department of Hematology, The Second Hospital of Hebei Medical University, Key Laboratory of Hematology, Shijiazhuang, China
| | - Ziyuan Nie
- Department of Hematology, The Second Hospital of Hebei Medical University, Key Laboratory of Hematology, Shijiazhuang, China
| | - Xiaoyan Zhang
- Department of Hematology, The Second Hospital of Hebei Medical University, Key Laboratory of Hematology, Shijiazhuang, China
| | - Yaqiong Lu
- Department of Hematology, The Second Hospital of Hebei Medical University, Key Laboratory of Hematology, Shijiazhuang, China
| | - Yuxia Pan
- Department of Hematology, The Second Hospital of Hebei Medical University, Key Laboratory of Hematology, Shijiazhuang, China
| | - Xingzhe Wang
- Department of Hematology, The Second Hospital of Hebei Medical University, Key Laboratory of Hematology, Shijiazhuang, China
| | - Jianmin Luo
- Department of Hematology, The Second Hospital of Hebei Medical University, Key Laboratory of Hematology, Shijiazhuang, China
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16
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Wu Y, Guo Q, Ju X, Hu Z, Xia L, Deng Y, Zhao P, Zhang M, Shao Y, Huang S, He X, Wen H, Wu X. HNRNPH1-stabilized LINC00662 promotes ovarian cancer progression by activating the GRP78/p38 pathway. Oncogene 2021; 40:4770-4782. [PMID: 34148056 PMCID: PMC8298204 DOI: 10.1038/s41388-021-01884-5] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2020] [Revised: 05/23/2021] [Accepted: 06/01/2021] [Indexed: 02/05/2023]
Abstract
Numerous studies suggest an important role for copy number alterations (CNAs) in cancer progression. However, CNAs of long intergenic noncoding RNAs (lincRNAs) in ovarian cancer (OC) and their potential functions have not been fully investigated. Here, based on analysis of The Cancer Genome Atlas (TCGA) database, we identified in this study an oncogenic lincRNA termed LINC00662 that exhibited a significant correlation between its CNA and its increased expression. LINC00662 overexpression is highly associated with malignant features in OC patients and is a prognostic indicator. LINC00662 significantly promotes OC cell proliferation and metastasis in vitro and in vivo. Mechanistically, LINC00662 is stabilized by heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1). Moreover, LINC00662 exerts oncogenic effects by interacting with glucose-regulated protein 78 (GRP78) and preventing its ubiquitination in OC cells, leading to activation of the oncogenic p38 MAPK signaling pathway. Taken together, our results define an oncogenic role for LINC00662 in OC progression mediated via GRP78/p38 signaling, with potential implications regarding therapeutic targets for OC.
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Affiliation(s)
- Yong Wu
- Department of Gynecologic Oncology, Fudan University Shanghai Cancer Center, Shanghai, 200032, China
- Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China
| | - Qinhao Guo
- Department of Gynecologic Oncology, Fudan University Shanghai Cancer Center, Shanghai, 200032, China
- Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China
| | - Xingzhu Ju
- Department of Gynecologic Oncology, Fudan University Shanghai Cancer Center, Shanghai, 200032, China
- Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China
| | - Zhixiang Hu
- Fudan University Shanghai Cancer Center, Key Laboratory of Medical Epigenetics and Metabolism, Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China
- Cancer Institute, Fudan University Shanghai Cancer Center, Shanghai, 200032, China
| | - Lingfang Xia
- Department of Gynecologic Oncology, Fudan University Shanghai Cancer Center, Shanghai, 200032, China
- Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China
| | - Yu Deng
- Department of Pathology, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 310000, China
| | - Ping Zhao
- Department of Pathology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, 200001, China
| | - Meng Zhang
- Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China
- Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, 200032, China
| | - Yang Shao
- Cancer Institute, Fudan University Shanghai Cancer Center, Shanghai, 200032, China
- Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai, 200032, China
| | - Shenglin Huang
- Fudan University Shanghai Cancer Center, Key Laboratory of Medical Epigenetics and Metabolism, Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China
- Cancer Institute, Fudan University Shanghai Cancer Center, Shanghai, 200032, China
| | - Xianghuo He
- Fudan University Shanghai Cancer Center, Key Laboratory of Medical Epigenetics and Metabolism, Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, China
- Cancer Institute, Fudan University Shanghai Cancer Center, Shanghai, 200032, China
| | - Hao Wen
- Department of Gynecologic Oncology, Fudan University Shanghai Cancer Center, Shanghai, 200032, China.
- Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China.
| | - Xiaohua Wu
- Department of Gynecologic Oncology, Fudan University Shanghai Cancer Center, Shanghai, 200032, China.
- Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China.
- Cancer Institute, Fudan University Shanghai Cancer Center, Shanghai, 200032, China.
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Dou Z, Zhao D, Chen X, Xu C, Jin X, Zhang X, Wang Y, Xie X, Li Q, Di C, Zhang H. Aberrant Bcl-x splicing in cancer: from molecular mechanism to therapeutic modulation. J Exp Clin Cancer Res 2021; 40:194. [PMID: 34118966 PMCID: PMC8196531 DOI: 10.1186/s13046-021-02001-w] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2021] [Accepted: 05/30/2021] [Indexed: 12/13/2022] Open
Abstract
Bcl-x pre-mRNA splicing serves as a typical example to study the impact of alternative splicing in the modulation of cell death. Dysregulation of Bcl-x apoptotic isoforms caused by precarious equilibrium splicing is implicated in genesis and development of multiple human diseases, especially cancers. Exploring the mechanism of Bcl-x splicing and regulation has provided insight into the development of drugs that could contribute to sensitivity of cancer cells to death. On this basis, we review the multiple splicing patterns and structural characteristics of Bcl-x. Additionally, we outline the cis-regulatory elements, trans-acting factors as well as epigenetic modifications involved in the splicing regulation of Bcl-x. Furthermore, this review highlights aberrant splicing of Bcl-x involved in apoptosis evade, autophagy, metastasis, and therapy resistance of various cancer cells. Last, emphasis is given to the clinical role of targeting Bcl-x splicing correction in human cancer based on the splice-switching oligonucleotides, small molecular modulators and BH3 mimetics. Thus, it is highlighting significance of aberrant splicing isoforms of Bcl-x as targets for cancer therapy.
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Affiliation(s)
- Zhihui Dou
- Department of Heavy Ion Radiation Medicine, Bio-Medical Research Center, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, 730000, China
- Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou, 730000, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 101408, China
- School of Nuclear Science and Technology, University of Chinese Academy of Sciences, Beijing, 101408, China
| | - Dapeng Zhao
- Department of Heavy Ion Radiation Medicine, Bio-Medical Research Center, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, 730000, China
- Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou, 730000, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 101408, China
- School of Nuclear Science and Technology, University of Chinese Academy of Sciences, Beijing, 101408, China
| | - Xiaohua Chen
- Department of Heavy Ion Radiation Medicine, Bio-Medical Research Center, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, 730000, China
- Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou, 730000, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 101408, China
- School of Nuclear Science and Technology, University of Chinese Academy of Sciences, Beijing, 101408, China
| | - Caipeng Xu
- Department of Heavy Ion Radiation Medicine, Bio-Medical Research Center, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, 730000, China
- Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou, 730000, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 101408, China
- School of Nuclear Science and Technology, University of Chinese Academy of Sciences, Beijing, 101408, China
| | - Xiaodong Jin
- Department of Heavy Ion Radiation Medicine, Bio-Medical Research Center, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, 730000, China
- Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou, 730000, China
| | - Xuetian Zhang
- Department of Heavy Ion Radiation Medicine, Bio-Medical Research Center, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, 730000, China
- Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou, 730000, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 101408, China
- School of Nuclear Science and Technology, University of Chinese Academy of Sciences, Beijing, 101408, China
| | - Yupei Wang
- Medical Genetics Center of Gansu Maternal and Child Health Care Center, Lanzhou, 730000, China
| | - Xiaodong Xie
- School of Basic Medical Sciences, Lanzhou University, Lanzhou, 730000, China
| | - Qiang Li
- Department of Heavy Ion Radiation Medicine, Bio-Medical Research Center, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, 730000, China
- Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou, 730000, China
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 101408, China
- School of Nuclear Science and Technology, University of Chinese Academy of Sciences, Beijing, 101408, China
- Advanced Energy Science and Technology Guangdong Laboratory, Huizhou, 516029, China
| | - Cuixia Di
- Department of Heavy Ion Radiation Medicine, Bio-Medical Research Center, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, 730000, China.
- Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou, 730000, China.
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 101408, China.
- School of Nuclear Science and Technology, University of Chinese Academy of Sciences, Beijing, 101408, China.
- Advanced Energy Science and Technology Guangdong Laboratory, Huizhou, 516029, China.
| | - Hong Zhang
- Department of Heavy Ion Radiation Medicine, Bio-Medical Research Center, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou, 730000, China.
- Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou, 730000, China.
- College of Life Sciences, University of Chinese Academy of Sciences, Beijing, 101408, China.
- School of Nuclear Science and Technology, University of Chinese Academy of Sciences, Beijing, 101408, China.
- Advanced Energy Science and Technology Guangdong Laboratory, Huizhou, 516029, China.
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18
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Choi N, Liu Y, Oh J, Ha J, Ghigna C, Zheng X, Shen H. Relative strength of 5’ splice-site strength defines functions of SRSF2 and SRSF6 in alternative splicing of Bcl-x pre-mRNA. BMB Rep 2021. [PMID: 33050987 PMCID: PMC8016662 DOI: 10.5483/bmbrep.2021.54.3.170] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
Bcl-x, a member of the Bcl-2 family, plays a key role in apoptosis. Alternative splicing of Bcl-x pre-mRNA through alternative 5’ splice-site selection produces an anti-apoptotic mRNA isoform that includes exon 2b and a pro-apoptotic Bcl-x mRNA isoform that excludes exon 2b. Here we used Bcl-x minigene and identified SRSF2 and SRSF6 as two regulatory factors of 5’ splice-site selection of Bcl-x pre-mRNA. We selected binding clusters closer to 5’ splice-sites from multiple potential binding sites of SRSF2 and SRSF6 to perform loss of functions analysis through site-directed mutagenesis. Our results demonstrated that these mutations did not abolish regulatory functions of SRSF2 or SRSF6, indicating that a single binding motif or a cluster was not a functional target of these proteins in Bcl-x pre-mRNA splicing. Random deletion mutagenesis did not disrupt the role of SRSF2 and SRSF6. Importantly, mutagenesis of 5’ splice-site to a conserved or a weaker score demonstrated that the weaker strength of the target 5’ splice-site or higher strength of the other 5’ splice-site strength limited the role of SRSF2 and SRSF6 in 5’ splice-site activation.
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Affiliation(s)
- Namjeong Choi
- School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 61005, Korea
| | - Yongchao Liu
- School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 61005, Korea
| | - Jagyeong Oh
- School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 61005, Korea
| | - Jiyeon Ha
- School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 61005, Korea
| | - Claudia Ghigna
- Institute of Molecular Genetics “Luigi Luca Cavalli-Sforza”, National Research Council, Pavia 27100, Italy
| | - Xuexiu Zheng
- School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 61005, Korea
| | - Haihong Shen
- School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 61005, Korea
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19
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Qin J, Autexier C. Regulation of human telomerase RNA biogenesis and localization. RNA Biol 2021; 18:305-315. [PMID: 32813614 PMCID: PMC7954027 DOI: 10.1080/15476286.2020.1809196] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2020] [Revised: 08/03/2020] [Accepted: 08/08/2020] [Indexed: 12/16/2022] Open
Abstract
Maintenance of telomeres is essential for genome integrity and replicative capacity in eukaryotic cells. Telomerase, the ribonucleoprotein complex that catalyses telomere synthesis is minimally composed of a reverse transcriptase and an RNA component. The sequence and structural domains of human telomerase RNA (hTR) have been extensively characterized, while the regulation of hTR transcription, maturation, and localization, is not fully understood. Here, we provide an up-to-date review of hTR, with an emphasis on current breakthroughs uncovering the mechanisms of hTR maturation and localization.
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Affiliation(s)
- Jian Qin
- Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada
- Jewish General Hospital, Lady Davis Institute, Montreal, Quebec, Canada
| | - Chantal Autexier
- Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada
- Jewish General Hospital, Lady Davis Institute, Montreal, Quebec, Canada
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20
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Lago S, Nadai M, Ruggiero E, Tassinari M, Marušič M, Tosoni B, Frasson I, Cernilogar FM, Pirota V, Doria F, Plavec J, Schotta G, Richter SN. The MDM2 inducible promoter folds into four-tetrad antiparallel G-quadruplexes targetable to fight malignant liposarcoma. Nucleic Acids Res 2021; 49:847-863. [PMID: 33410915 PMCID: PMC7826256 DOI: 10.1093/nar/gkaa1273] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2020] [Revised: 12/18/2020] [Accepted: 12/21/2020] [Indexed: 02/06/2023] Open
Abstract
Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis.
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Affiliation(s)
- Sara Lago
- Department of Molecular Medicine, University of Padua, via A. Gabelli 63, 35121 Padua, Italy
| | - Matteo Nadai
- Department of Molecular Medicine, University of Padua, via A. Gabelli 63, 35121 Padua, Italy
| | - Emanuela Ruggiero
- Department of Molecular Medicine, University of Padua, via A. Gabelli 63, 35121 Padua, Italy
| | - Martina Tassinari
- Department of Molecular Medicine, University of Padua, via A. Gabelli 63, 35121 Padua, Italy
| | - Maja Marušič
- Slovenian NMR center, National Institute of Chemistry, Hajdrihova, 19, Ljubljana SI-1000, Slovenia
| | - Beatrice Tosoni
- Department of Molecular Medicine, University of Padua, via A. Gabelli 63, 35121 Padua, Italy
| | - Ilaria Frasson
- Department of Molecular Medicine, University of Padua, via A. Gabelli 63, 35121 Padua, Italy
| | - Filippo M Cernilogar
- Division of Molecular Biology, Biomedical Center, Faculty of Medicine, LMU Munich, Germany
| | - Valentina Pirota
- Department of Chemistry, University of Pavia, V. le Taramelli 10, 27100, Pavia, Italy
| | - Filippo Doria
- Department of Chemistry, University of Pavia, V. le Taramelli 10, 27100, Pavia, Italy
| | - Janez Plavec
- Slovenian NMR center, National Institute of Chemistry, Hajdrihova, 19, Ljubljana SI-1000, Slovenia
| | - Gunnar Schotta
- Division of Molecular Biology, Biomedical Center, Faculty of Medicine, LMU Munich, Germany
| | - Sara N Richter
- Department of Molecular Medicine, University of Padua, via A. Gabelli 63, 35121 Padua, Italy
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21
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PKC Regulates YAP Expression through Alternative Splicing of YAP 3'UTR Pre-mRNA by hnRNP F. Int J Mol Sci 2021; 22:ijms22020694. [PMID: 33445676 PMCID: PMC7828143 DOI: 10.3390/ijms22020694] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2020] [Revised: 01/07/2021] [Accepted: 01/09/2021] [Indexed: 01/14/2023] Open
Abstract
The Yes-associated protein (YAP) is a transcriptional co-activator that plays critical roles in organ development and tumorigenesis, and is verified to be inhibited by the Hippo signaling pathway. In the present study, we show that the YAP 3′UTR is alternatively spliced to generate a novel 950 bp 3′UTR mRNA from the full length 3′UTR region (3483 bp) in human cancer cells. The ratio of full length 3′UTR YAP mRNA to alternatively spliced 3′UTR YAP mRNA is up-regulated by exposure of the cells to PKC inhibitor chelerythrine chloride. Further study using luciferase reporter assay showed that the expression of the alternatively spliced 3′UTR mRNA is much lower compared with the full length 3′UTR mRNA, suggesting that alternatively spliced 3′UTR YAP mRNA may have a shorter half-life than full length 3′UTR mRNA. Interestingly, PKC represses YAP 3′UTR–mediated mRNA stability is dependent on a splicing factor, hnRNP F. Activation of PKC induces nuclear translocation of cytosolic hnRNP F. Ectopic expression of hnRNP F enhances YAP 3′UTR splicing. Our results suggest that hnRNP F regulates YAP 3′UTR-mediated mRNA stability in an alternative splicing-dependent manner, and PKC regulated YAP expression is dependent on nuclear translocation of hnRNP F in human cancer cell lines.
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22
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Wall ML, Bera A, Wong FK, Lewis SM. Cellular stress orchestrates the localization of hnRNP H to stress granules. Exp Cell Res 2020; 394:112111. [PMID: 32473225 DOI: 10.1016/j.yexcr.2020.112111] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2020] [Revised: 05/19/2020] [Accepted: 05/21/2020] [Indexed: 12/01/2022]
Abstract
Heterogeneous nuclear ribonucleoprotein (hnRNP) H is a member of hnRNP H/F protein subfamily of hnRNPs that regulate the maturation and post-transcriptional processing of pre-mRNA. As a component of an mRNA export complex, hnRNP H shuttles mature mRNA from the nucleus to the cytoplasm. Although hnRNP H is primarily a nuclear protein, it can accumulate in the cytoplasm in certain tissues and cell types; however, the physiological relevance of hnRNP H cytoplasmic accumulation is unknown. Here we show that under cellular stress hnRNP H accumulates in the cytoplasm and is required for efficient recovery from cellular stress. Moreover, we find that cytoplasmic hnRNP H localizes to stress granules and that the RRM3 domain of hnRNP H is necessary for this localization. Together, our results demonstrate that hnRNP H accumulates in the cytoplasm under cellular stress and is recruited to stress granules.
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Affiliation(s)
- Michael L Wall
- Atlantic Cancer Research Institute, Moncton, New Brunswick, Canada
| | - Amit Bera
- Atlantic Cancer Research Institute, Moncton, New Brunswick, Canada
| | - Florence K Wong
- Atlantic Cancer Research Institute, Moncton, New Brunswick, Canada
| | - Stephen M Lewis
- Atlantic Cancer Research Institute, Moncton, New Brunswick, Canada; Department of Chemistry & Biochemistry, Université de Moncton, Moncton, New Brunswick, Canada; Senior Scientist, Beatrice Hunter Cancer Research Institute, Canada.
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23
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Xu C, Xie N, Su Y, Sun Z, Liang Y, Zhang N, Liu D, Jia S, Xing X, Han L, Li G, Tong T, Chen J. HnRNP F/H associate with hTERC and telomerase holoenzyme to modulate telomerase function and promote cell proliferation. Cell Death Differ 2019; 27:1998-2013. [PMID: 31863069 PMCID: PMC7244589 DOI: 10.1038/s41418-019-0483-6] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2019] [Revised: 12/09/2019] [Accepted: 12/10/2019] [Indexed: 12/16/2022] Open
Abstract
Human telomerase RNA component hTERC comprises multiple motifs that contribute to hTERC biogenesis, holoenzyme activity, and enzyme recruitment to telomeres. hTERC contains several guanine tracts (G-tracts) at its 5′-end, but its associated proteins and potential roles in telomerase function are still poorly understood. The heterogeneous nuclear ribonucleoproteins F, H1, and H2 (hnRNP F/H) are splicing factors that preferentially bind to poly(G)-rich sequences RNA. Here, we demonstrate that hnRNP F/H associate with both hTERC and telomerase holoenzyme to regulate telomerase activity. We reveal hnRNP F/H bind to the 5′-end region of hTERC in vitro and in vivo, and identify the first three G-tracts of hTERC and qRRM1 domain of hnRNP F/H are required for their interaction. Furthermore, hnRNP F/H also directly interact with telomerase holoenzyme. Functionally, we show that hnRNP F/H plays important roles in modulating telomerase activity and telomere length. Moreover, hnRNP F/H deletion greatly impair cancer and stem cell proliferation, and induce stem cell senescence, while hnRNP F/H overexpression delay stem cell senescence. Collectively, our findings unveil a novel role of hnRNP F/H as the binding partners of hTERC and telomerase holoenzyme to regulate telomerase function.
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Affiliation(s)
- Chenzhong Xu
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Nan Xie
- Department of Physiology and Pathophysiology, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Yuanyuan Su
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Zhaomeng Sun
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Yao Liang
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Na Zhang
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Doudou Liu
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Shuqin Jia
- Department of Molecular Diagnostics, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute, Beijing, 100142, China
| | - Xiaofang Xing
- Department of Molecular Diagnostics, Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Peking University Cancer Hospital & Institute, Beijing, 100142, China
| | - Limin Han
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Guodong Li
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Tanjun Tong
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China
| | - Jun Chen
- Peking University Research Center on Aging, Beijing Key Laboratory of Protein Posttranslational Modifications and Cell Function, Department of Biochemistry and Molecular Biology, Department of Integration of Chinese and Western Medicine, School of Basic Medical Science, Peking University, Beijing, 100191, China.
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24
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Neckles C, Boer RE, Aboreden N, Cross AM, Walker RL, Kim BH, Kim S, Schneekloth JS, Caplen NJ. HNRNPH1-dependent splicing of a fusion oncogene reveals a targetable RNA G-quadruplex interaction. RNA (NEW YORK, N.Y.) 2019; 25:1731-1750. [PMID: 31511320 PMCID: PMC6859848 DOI: 10.1261/rna.072454.119] [Citation(s) in RCA: 34] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/25/2019] [Accepted: 09/08/2019] [Indexed: 05/05/2023]
Abstract
The primary oncogenic event in ∼85% of Ewing sarcomas is a chromosomal translocation that generates a fusion oncogene encoding an aberrant transcription factor. The exact genomic breakpoints within the translocated genes, EWSR1 and FLI1, vary; however, in EWSR1, breakpoints typically occur within introns 7 or 8. We previously found that in Ewing sarcoma cells harboring EWSR1 intron 8 breakpoints, the RNA-binding protein HNRNPH1 facilitates a splicing event that excludes EWSR1 exon 8 from the EWS-FLI1 pre-mRNA to generate an in-frame mRNA. Here, we show that the processing of distinct EWS-FLI1 pre-mRNAs by HNRNPH1, but not other homologous family members, resembles alternative splicing of transcript variants of EWSR1 We demonstrate that HNRNPH1 recruitment is driven by guanine-rich sequences within EWSR1 exon 8 that have the potential to fold into RNA G-quadruplex structures. Critically, we demonstrate that an RNA mimetic of one of these G-quadruplexes modulates HNRNPH1 binding and induces a decrease in the growth of an EWSR1 exon 8 fusion-positive Ewing sarcoma cell line. Finally, we show that EWSR1 exon 8 fusion-positive cell lines are more sensitive to treatment with the pan-quadruplex binding molecule, pyridostatin (PDS), than EWSR1 exon 8 fusion-negative lines. Also, the treatment of EWSR1 exon 8 fusion-positive cells with PDS decreases EWS-FLI1 transcriptional activity, reversing the transcriptional deregulation driven by EWS-FLI1. Our findings illustrate that modulation of the alternative splicing of EWS-FLI1 pre-mRNA is a novel strategy for future therapeutics against the EWSR1 exon 8 containing fusion oncogenes present in a third of Ewing sarcoma.
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Affiliation(s)
- Carla Neckles
- Functional Genetics Section, Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, USA
| | - Robert E Boer
- Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA
| | - Nicholas Aboreden
- Functional Genetics Section, Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, USA
| | - Allison M Cross
- Functional Genetics Section, Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, USA
| | - Robert L Walker
- Molecular Genetics Section, Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, USA
| | - Bong-Hyun Kim
- CCR Collaborative Bioinformatics Resource, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., Frederick, Maryland 21702, USA
| | - Suntae Kim
- Functional Genetics Section, Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, USA
| | - John S Schneekloth
- Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, Maryland 21702, USA
| | - Natasha J Caplen
- Functional Genetics Section, Genetics Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland 20892, USA
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25
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Stevens M, Oltean S. Modulation of the Apoptosis Gene Bcl-x Function Through Alternative Splicing. Front Genet 2019; 10:804. [PMID: 31552099 PMCID: PMC6743414 DOI: 10.3389/fgene.2019.00804] [Citation(s) in RCA: 83] [Impact Index Per Article: 13.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2019] [Accepted: 07/31/2019] [Indexed: 01/09/2023] Open
Abstract
Apoptosis plays a vital role in cell homeostasis during development and disease. Bcl-x, a member of the Bcl-2 family of proteins, is a mitochondrial transmembrane protein that functions to regulate the intrinsic apoptosis pathway. An alternative splicing (AS) event in exon 2 of Bcl-x results in two isoforms of Bcl-x with antagonistic effects on cell survival: Bcl-xL (long isoform), which is anti-apoptotic, and Bcl-xS (short isoform), which is pro-apoptotic. Bcl-xL is the most abundant Bcl-x protein and functions to inhibit apoptosis by a number of different mechanisms including inhibition of Bax. In contrast, Bcl-xS can directly bind to and inhibit the anti-apoptotic Bcl-xL and Bcl-2 proteins, resulting in the release of the pro-apoptotic Bak. There are multiple splice factors and signaling pathways that influence the Bcl-xL/Bcl-xS splicing ratio, including serine/arginine-rich (SR) proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), transcription factors, and cytokines. Dysregulation of the AS of Bcl-x has been implicated in cancer and diabetes. In cancer, the upregulation of Bcl-xL expression in tumor cells can result in resistance to chemotherapeutic agents. On the other hand, dysregulation of Bcl-x AS to promote Bcl-xS expression has been shown to be detrimental to pancreatic β-cells in diabetes, resulting in β-cell apoptosis. Therefore, manipulation of the splice factor, transcription factor, and signaling pathways that modulate this splicing event is fast emerging as a therapeutic avenue in the treatment of cancer and diabetes.
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Affiliation(s)
- Megan Stevens
- Institute of Biomedical and Clinical Science, Medical School, College of Medicine and Health, University of Exeter, Exeter, United Kingdom
| | - Sebastian Oltean
- Institute of Biomedical and Clinical Science, Medical School, College of Medicine and Health, University of Exeter, Exeter, United Kingdom
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26
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Weldon C, Dacanay JG, Gokhale V, Boddupally PVL, Behm-Ansmant I, Burley GA, Branlant C, Hurley LH, Dominguez C, Eperon IC. Specific G-quadruplex ligands modulate the alternative splicing of Bcl-X. Nucleic Acids Res 2019; 46:886-896. [PMID: 29156002 PMCID: PMC5778605 DOI: 10.1093/nar/gkx1122] [Citation(s) in RCA: 50] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2017] [Accepted: 10/26/2017] [Indexed: 12/23/2022] Open
Abstract
Sequences with the potential to form RNA G-quadruplexes (G4s) are common in mammalian introns, especially in the proximity of the 5′ splice site (5′SS). However, the difficulty of demonstrating that G4s form in pre-mRNA in functional conditions has meant that little is known about their effects or mechanisms of action. We have shown previously that two G4s form in Bcl-X pre-mRNA, one close to each of the two alternative 5′SS. If these G4s affect splicing but are in competition with other RNA structures or RNA binding proteins, then ligands that stabilize them would increase the proportion of Bcl-X pre-mRNA molecules in which either or both G4s had formed, shifting Bcl-X splicing. We show here that a restricted set of G4 ligands do affect splicing, that their activity and specificity are strongly dependent on their structures and that they act independently at the two splice sites. One of the ligands, the ellipticine GQC-05, antagonizes the major 5′SS that expresses the anti-apoptotic isoform of Bcl-X and activates the alternative 5′SS that expresses a pro-apoptotic isoform. We propose mechanisms that would account for these see-saw effects and suggest that these effects contribute to the ability of GQC-05 to induce apoptosis.
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Affiliation(s)
- Carika Weldon
- Leicester Institute of Structural & Chemical Biology and Department of Molecular & Cell Biology, University of Leicester, Leicester LE1 7RH, UK
| | - Justine G Dacanay
- Leicester Institute of Structural & Chemical Biology and Department of Molecular & Cell Biology, University of Leicester, Leicester LE1 7RH, UK
| | - Vijay Gokhale
- College of Pharmacy and College of Pharmacy and BIO5 Institute, University of Arizona, Tucson, AZ 85721, USA
| | - Peda Venkat L Boddupally
- Fluoroorganic Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, Telangana 500 007, India
| | - Isabelle Behm-Ansmant
- IMoPA (Ingénierie Moléculaire et Physiopathologie Articulaire), UMR 7365 CNRS-UL, Biopôle de l'Université de Lorraine, 9 Avenue de la Forêt de Haye, 54505 Vandoeuvre-lès-Nancy, France
| | - Glenn A Burley
- Department of Pure and Applied Chemistry, University of Strathclyde, Glasgow G1 1XL, UK
| | - Christiane Branlant
- IMoPA (Ingénierie Moléculaire et Physiopathologie Articulaire), UMR 7365 CNRS-UL, Biopôle de l'Université de Lorraine, 9 Avenue de la Forêt de Haye, 54505 Vandoeuvre-lès-Nancy, France
| | - Laurence H Hurley
- College of Pharmacy and College of Pharmacy and BIO5 Institute, University of Arizona, Tucson, AZ 85721, USA.,Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA
| | - Cyril Dominguez
- Leicester Institute of Structural & Chemical Biology and Department of Molecular & Cell Biology, University of Leicester, Leicester LE1 7RH, UK
| | - Ian C Eperon
- Leicester Institute of Structural & Chemical Biology and Department of Molecular & Cell Biology, University of Leicester, Leicester LE1 7RH, UK
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27
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Bartys N, Kierzek R, Lisowiec-Wachnicka J. The regulation properties of RNA secondary structure in alternative splicing. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2019; 1862:194401. [PMID: 31323437 DOI: 10.1016/j.bbagrm.2019.07.002] [Citation(s) in RCA: 30] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/29/2019] [Accepted: 07/09/2019] [Indexed: 11/30/2022]
Abstract
The RNA secondary structure is important for many functional processes in the cell. The secondary and tertiary structures of cellular RNAs are essential for the activity of these molecules in processes such as transcription, splicing, translation, and localization. New high-throughput analytical methods, including next generation sequencing, have allowed for the in-depth characterization of the 'RNA structurome': a new term describing how the RNA structure controls the activity of RNA by itself and how it regulates the expression of genes. In this review, we present many examples of the influence of structural motifs of RNA, long range interactions and global RNA structure on the alternative splicing processes. This article is part of a Special Issue entitled: RNA structure and splicing regulation edited by Francisco Baralle, Ravindra Singh and Stefan Stamm.
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Affiliation(s)
- Natalia Bartys
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznań, Poland
| | - Ryszard Kierzek
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznań, Poland
| | - Jolanta Lisowiec-Wachnicka
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznań, Poland.
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Pokhriyal R, Hariprasad R, Kumar L, Hariprasad G. Chemotherapy Resistance in Advanced Ovarian Cancer Patients. BIOMARKERS IN CANCER 2019; 11:1179299X19860815. [PMID: 31308780 PMCID: PMC6613062 DOI: 10.1177/1179299x19860815] [Citation(s) in RCA: 180] [Impact Index Per Article: 30.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/13/2019] [Accepted: 05/08/2019] [Indexed: 12/26/2022]
Abstract
Ovarian cancer is the seventh most common gynaecologic malignancy seen in women. Majority of the patients with ovarian cancer are diagnosed at the advanced stage making prognosis poor. The standard management of advanced ovarian cancer includes tumour debulking surgery followed by chemotherapy. Various types of chemotherapeutic regimens have been used to treat advanced ovarian cancer, but the most promising and the currently used standard first-line treatment is carboplatin and paclitaxel. Despite improved clinical response and survival to this combination of chemotherapy, numerous patients either undergo relapse or succumb to the disease as a result of chemotherapy resistance. To understand this phenomenon at a cellular level, various macromolecules such as DNA, messenger RNA and proteins have been developed as biomarkers for chemotherapy response. This review comprehensively summarizes the problem that pertains to chemotherapy resistance in advanced ovarian cancer and provides a good overview of the various biomarkers that have been developed in this field.
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Affiliation(s)
- Ruchika Pokhriyal
- Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India
| | - Roopa Hariprasad
- Division of Clinical Oncology, National Institute of Cancer Prevention and Research, Noida, India
| | - Lalit Kumar
- Department of Medical Oncology, All India Institute of Medical Sciences, New Delhi, India
| | - Gururao Hariprasad
- Department of Biophysics, All India Institute of Medical Sciences, New Delhi, India
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29
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Li F, Zhao H, Su M, Xie W, Fang Y, Du Y, Yu Z, Hou L, Tan W. HnRNP-F regulates EMT in bladder cancer by mediating the stabilization of Snail1 mRNA by binding to its 3' UTR. EBioMedicine 2019; 45:208-219. [PMID: 31221586 PMCID: PMC6642227 DOI: 10.1016/j.ebiom.2019.06.017] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2019] [Revised: 06/04/2019] [Accepted: 06/11/2019] [Indexed: 12/11/2022] Open
Abstract
Background Heterogeneous nuclear ribonucleoprotein F (hnRNP-F) has been implicated in multiple cancers, suggesting its role in tumourigenesis, but the potential oncogenic role and mechanism of hnRNP-F in bladder cancer (BC) remain incompletely understood. Methods HnRNP-F was identified by proteomic methods. A correlation of hnRNP-F expression with prognosis was analysed in 103 BC patients. Then, we applied in vitro and in vivo methods to reveal the behaviours of hnRNP-F in BC tumourigenesis. Furthermore, the interaction between hnRNP-F and Snail1 mRNA was examined by RNA immunoprecipitation (RIP), and Snail1 mRNA stability was measured after treatment with actinomycin D. Finally, the binding domain between hnRNP-F and Snail1 mRNA was verified by constructing Snail1 mRNA truncations and mutants. Finding HnRNP-F is significantly upregulated in BC tissue, and its increased expression is associated with a poor prognosis in BC patients. HnRNP-F is necessary for tumour growth, inducing epithelial-mesenchymal transition (EMT) and metastasis in BC. The changes in Snail1 expression were positively correlated with hnRNP-F at both the mRNA and protein levels when hnRNP-F was silenced or enhanced, suggesting that Snail1 is likely a downstream target of hnRNP-F that mediates its effects on enhancing invasion, metastasis and EMT in BC. The overexpression of hnRNP-F caused an increase in the stability of Snail1 mRNA. Our RNA chip analysis revealed that hnRNP-F could combine with Snail1 mRNA, and we further demonstrated that hnRNP-F could directly bind to the 3′ untranslated region (3′ UTR) of Snail1 mRNA to enhance its stability. Interpretation Our findings suggest that hnRNP-F mediates the stabilization of Snail1 mRNA by binding to its 3′ UTR, subsequently regulating EMT.
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Affiliation(s)
- Fei Li
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, PR China
| | - Hongfan Zhao
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, PR China
| | - Mingqiang Su
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, PR China
| | - Weiwei Xie
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, PR China
| | - Yunze Fang
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, PR China
| | - Yuejun Du
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, PR China
| | - Zhe Yu
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, PR China.
| | - Lina Hou
- Department of Healthy Management, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, PR China.
| | - Wanlong Tan
- Department of Urology, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong 510515, PR China.
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Hirata BKS, Pedroso AP, Machado MMF, Neto NIP, Perestrelo BO, de Sá RDCC, Alonso-Vale MIC, Nogueira FN, Oyama LM, Ribeiro EB, Tashima AK, Telles MM. Ginkgo biloba Extract Modulates the Retroperitoneal Fat Depot Proteome and Reduces Oxidative Stress in Diet-Induced Obese Rats. Front Pharmacol 2019; 10:686. [PMID: 31258482 PMCID: PMC6587378 DOI: 10.3389/fphar.2019.00686] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2019] [Accepted: 05/27/2019] [Indexed: 12/18/2022] Open
Abstract
The rapid increase in the number of individuals with obesity, over the past four decades, is triggered by a number of complex interactions among factors. Despite the plethora of treatments available, side effects are commonly observed and, in this context, herbal medicines have been employed as an alternative form of therapy. Ginkgo biloba extract (GbE) has been described as a promising new pharmacological approach to treat obesity. In order to better comprehend the mechanisms involved with this potential effect, the present study evaluated the effects of GbE treatment on diet-induced obese rats, focusing on the proteome and the oxidative stress defense system of visceral adipose tissue. After 14 days treatment, GbE significantly modulated 25 proteins. Retroperitoneal adipose tissue of treated animals exhibited higher amounts of proteins associated with adipogenesis (decorin), carbon metabolism and mitochondrial function (citrate synthase), and a concomitant reduction in adipocyte hypertrophy. In parallel, GbE down-regulated proteins involved in oxidative stress (peroxiredoxin) and the inflammatory response (complement C3, mast cell protease 1, and Ig gamma-2B chain C region). Moreover, also related to oxidative stress defense, GbE stimulated catalase activity, reduced malondialdehyde levels (lipid peroxidation indicator), and increased lactoylglutathione lyase levels. It was concluded that GbE acts as an antioxidant agent, and improved the proteome profile and oxidative stress response in the adipose tissue of diet-induced obese rats.
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Affiliation(s)
- Bruna K S Hirata
- Department of Biological Sciences, Universidade Federal de São Paulo-UNIFESP, Diadema, Brazil
| | - Amanda P Pedroso
- Department of Physiology, Universidade Federal de São Paulo-UNIFESP, São Paulo, Brazil
| | - Meira M F Machado
- Department of Biological Sciences, Universidade Federal de São Paulo-UNIFESP, Diadema, Brazil
| | - Nelson I P Neto
- Department of Physiology, Universidade Federal de São Paulo-UNIFESP, São Paulo, Brazil
| | - Bruna O Perestrelo
- Department of Biomaterials and Oral Biology, School of Dentistry, Universidade de São Paulo-USP, São Paulo, Brazil
| | - Roberta D C C de Sá
- Department of Biological Sciences, Universidade Federal de São Paulo-UNIFESP, Diadema, Brazil
| | | | - Fernando N Nogueira
- Department of Biomaterials and Oral Biology, School of Dentistry, Universidade de São Paulo-USP, São Paulo, Brazil
| | - Lila M Oyama
- Department of Physiology, Universidade Federal de São Paulo-UNIFESP, São Paulo, Brazil
| | - Eliane B Ribeiro
- Department of Physiology, Universidade Federal de São Paulo-UNIFESP, São Paulo, Brazil
| | - Alexandre K Tashima
- Department of Biochemistry, Universidade Federal de São Paulo-UNIFESP, São Paulo, Brazil
| | - Monica M Telles
- Department of Biological Sciences, Universidade Federal de São Paulo-UNIFESP, Diadema, Brazil
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31
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Zong L, Hattori N, Yasukawa Y, Kimura K, Mori A, Seto Y, Ushijima T. LINC00162 confers sensitivity to 5-Aza-2'-deoxycytidine via modulation of an RNA splicing protein, HNRNPH1. Oncogene 2019; 38:5281-5293. [PMID: 30914798 DOI: 10.1038/s41388-019-0792-8] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2018] [Revised: 03/12/2019] [Accepted: 03/14/2019] [Indexed: 12/13/2022]
Abstract
DNA demethylation therapy is now expanding from hematological tumors to solid tumors. To exploit its maximum efficacy, long-term treatment is needed, and stratification of sensitive patients is critically important. Here, we identified a long non-coding RNA, LINC00162, as highly and frequently expressed in gastric cancer cell lines sensitive to 5-aza-2'-deoxycytidine (5-aza-dC). Knockdown of LINC00162 decreased the sensitivity while its overexpression increased the sensitivity. In vivo experiments also showed that LINC00162 overexpression increased the sensitivity. LINC00162 enhanced cell cycle arrest and apoptosis induced by 5-aza-dC, but did not affect its DNA demethylation effect. Mechanistically, LINC00162 interacted with an RNA splicing protein, HNRNPH1, and decreased splicing of an anti-apoptotic splicing variant, BCL-XL. LINC00162 may have translational value to predict patients who will respond to 5-aza-dC.
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Affiliation(s)
- Liang Zong
- Division of Epigenomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan
- Department of Gastrointestinal Surgery, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan
| | - Naoko Hattori
- Division of Epigenomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan
| | - Yoshimi Yasukawa
- Division of Epigenomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan
- Department of Gastrointestinal Surgery, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan
| | - Kana Kimura
- Division of Epigenomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan
| | - Akiko Mori
- Division of Epigenomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan
| | - Yasuyuki Seto
- Department of Gastrointestinal Surgery, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan
| | - Toshikazu Ushijima
- Division of Epigenomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan.
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BCL-2 family isoforms in apoptosis and cancer. Cell Death Dis 2019; 10:177. [PMID: 30792387 PMCID: PMC6384907 DOI: 10.1038/s41419-019-1407-6] [Citation(s) in RCA: 453] [Impact Index Per Article: 75.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2018] [Revised: 12/17/2018] [Accepted: 01/29/2019] [Indexed: 12/17/2022]
Abstract
The BCl-2 family has long been identified for its role in apoptosis. Following the initial discovery of BCL-2 in the context of B-cell lymphoma in the 1980s, a number of homologous proteins have since been identified. The members of the Bcl-2 family are designated as such due to their BCL-2 homology (BH) domains and involvement in apoptosis regulation. The BH domains facilitate the family members’ interactions with each other and can indicate pro- or anti-apoptotic function. Traditionally, these proteins are categorised into one of the three subfamilies; anti-apoptotic, BH3-only (pro-apoptotic), and pore-forming or ‘executioner’ (pro-apoptotic) proteins. Each of the BH3-only or anti-apoptotic proteins has a distinct pattern of activation, localisation and response to cell death or survival stimuli. All of these can vary across cell or stress types, or developmental stage, and this can cause the delineation of the roles of BCL-2 family members. Added to this complexity is the presence of relatively uncharacterised isoforms of many of the BCL-2 family members. There is a gap in our knowledge regarding the function of BCL-2 family isoforms. BH domain status is not always predictive or indicative of protein function, and several other important sequences, which can contribute to apoptotic activity have been identified. While therapeutic strategies targeting the BCL-2 family are constantly under development, it is imperative that we understand the molecules, which we are attempting to target. This review, discusses our current knowledge of anti-apoptotic BCL-2 family isoforms. With significant improvements in the potential for splicing therapies, it is important that we begin to understand the distinctions of the BCL-2 family, not limited to just the mechanisms of apoptosis control, but in their roles outside of apoptosis.
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Tyson-Capper A, Gautrey H. Regulation of Mcl-1 alternative splicing by hnRNP F, H1 and K in breast cancer cells. RNA Biol 2018; 15:1448-1457. [PMID: 30468106 PMCID: PMC6333436 DOI: 10.1080/15476286.2018.1551692] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2018] [Revised: 10/10/2018] [Accepted: 11/02/2018] [Indexed: 01/27/2023] Open
Abstract
Myeloid cell leukemia-1 (Mcl -1) is one of the most frequently amplified genes in cancer, and its overexpression is associated with poor prognosis and drug resistance. As a member of the Bcl-2 family it is involved in the control of the mitochondrial (intrinsic) cell death pathway. Alternative splicing of the (Mcl-1) gene results in the expression of two functionally distinct proteins, the anti-apoptotic Mcl-1L (exon 2 included) and the pro-apoptotic Mcl-1S (exon 2 skipped). Our data shows that transfecting siRNAs that target hnRNP K and the hnRNP F/H family result in a switch in splicing towards the pro-apoptotic Mcl-1S. Specific binding sites for these and other Mcl-1 splicing factors were investigated and identified by RNA immunoprecipitation and through construction of a Mcl-1 minigene construct. Moreover, this study shows up to a 30 fold change in the levels of Mcl-1S can be achieved through double and triple knockdowns of the most significant RNA binding proteins involved in Mcl-1 splicing, as well as activation of the mitochondrial cell death pathway. Targeting the splicing process of Mcl-1 along with other apoptotic regulators provides an exciting new therapeutic target in cancer cells, and may provide a way to overcome therapy resistance.
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Affiliation(s)
- Alison Tyson-Capper
- Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
| | - Hannah Gautrey
- Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, UK
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Chu WK, Hung LM, Hou CW, Chen JK. Heterogeneous ribonucleoprotein F regulates YAP expression via a G-tract in 3'UTR. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2018; 1862:12-24. [PMID: 30312683 DOI: 10.1016/j.bbagrm.2018.10.003] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/27/2018] [Revised: 10/05/2018] [Accepted: 10/06/2018] [Indexed: 10/28/2022]
Abstract
The Yes-associated protein (YAP) is a transcription coactivator that plays crucial roles in organ size control and tumorigenesis, and was demonstrated to be inhibited by the Hippo signaling pathway. To date, the molecular mechanisms regulating the expression of YAP in human cells remain unknown. In the present study, we found that hnRNP F and hnRNP U negatively regulate YAP expression. We also showed that downregulation of YAP expression by hnRNP F and hnRNP U was not at the transcriptional level. Knockdown of hnRNP F or hnRNP U increased YAP mRNA stability, suggesting the downregulation of YAP expression was by a post-transcriptional mechanism. A putative hnRNP F binding site was identified in the YAP 3'UTR at 685 to 698, and deletion of this putative hnRNP F element abolished the down-regulation effect of YAP mRNA stability by hnRNP F. Binding of the hnRNP F to the YAP 3'UTR was demonstrated by Cross-linked RNA Immunoprecipitation. mRNA stability is a possible secondary effect of alternative splicing or other nuclear process. Understanding the regulation of YAP expression would provide insights into the mechanisms underlying the maintenance of tissue size homeostasis and tumorigenesis.
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Affiliation(s)
- Wing-Keung Chu
- Department of Physiology, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan; Healthy and Aging Research Center, Chang Gung University, Taoyuan 333, Taiwan
| | - Li-Man Hung
- Healthy and Aging Research Center, Chang Gung University, Taoyuan 333, Taiwan; Department of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan
| | - Chun-Wei Hou
- Healthy and Aging Research Center, Chang Gung University, Taoyuan 333, Taiwan
| | - Jan-Kan Chen
- Department of Physiology, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan; Healthy and Aging Research Center, Chang Gung University, Taoyuan 333, Taiwan.
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35
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Zhu J, Zhu LWS, Yang JH, Xu YL, Wang C, Li ZY, Mao W, Lu DZ. Proteomic analysis of human umbilical vein endothelial cells exposed to PM2.5. J Zhejiang Univ Sci B 2018. [DOI: 10.1631/jzus.b1700103] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
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Multilevel Differential Control of Hormone Gene Expression Programs by hnRNP L and LL in Pituitary Cells. Mol Cell Biol 2018; 38:MCB.00651-17. [PMID: 29610151 PMCID: PMC5974433 DOI: 10.1128/mcb.00651-17] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2017] [Accepted: 03/22/2018] [Indexed: 12/20/2022] Open
Abstract
The pituitary-derived somatolactotrophe GH3 cells secrete both growth hormone (GH) and prolactin (PRL). We have found that the hnRNP L and L-like (LL) paralogs differentially regulate alternative splicing of genes in these cells. Here, we show that hnRNP L is essential for PRL only, but LL is essential for both PRL and GH production. Transcriptome-wide RNA sequencing (RNA-Seq) analysis indicates that they differentially control groups of hormone or hormone-related genes involved in hormone production/regulation at total transcript and alternative exon levels. Interestingly, hnRNP L also specifically binds and prevents the aberrant usage of a nonconserved CA-rich intron piece of Prl pre-mRNA transcripts, and many others involved in endocrine functions, to prevent mostly cryptic last exons and mRNA truncation. Essential for the full hnRNP L effect on specific exons is a proline-rich region that emerged during evolution in vertebrate hnRNP L only but not LL. Together, our data demonstrate that the hnRNP L and its paralog, LL, differentially control hormone gene expression programs at multiple levels, and hnRNP L in particular is critical for protecting the transcriptome from aberrant usage of intronic sequences. The multilevel differential control by hnRNPs likely tailors the transcriptome to help refine and safeguard the different gene expression programs for different hormones.
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Du J, Wang Q, Ziegler SF, Zhou B. FOXP3 interacts with hnRNPF to modulate pre-mRNA alternative splicing. J Biol Chem 2018; 293:10235-10244. [PMID: 29773655 DOI: 10.1074/jbc.ra117.001349] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2017] [Revised: 05/01/2018] [Indexed: 12/13/2022] Open
Abstract
FOXP3 promotes the development and function of regulatory T cells mainly through regulating the transcription of target genes. RNA alternative splicing has been implicated in a wide range of physiological and pathophysiological processes. We report here that FOXP3 associates with heterogeneous nuclear ribonucleoprotein (hnRNP) F through the exon 2-encoded region of FOXP3 and the second quasi-RNA recognition motif (qRRM) of hnRNPF. FOXP3 represses the ability of hnRNPF to bind to its target pre-mRNA and thus modulates RNA alternative splicing. Furthermore, overexpression of mouse hnRNPF in in vitro-differentiated regulatory T cells (Tregs) reduced their suppressive function. Thus, our studies identify a novel mechanism by which FOXP3 regulates mRNA alternative splicing to modulate the function of regulatory T cells.
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Affiliation(s)
- Jianguang Du
- From the Wells Center for Pediatric Research and Department of Pediatrics and
| | - Qun Wang
- From the Wells Center for Pediatric Research and Department of Pediatrics and
| | - Steven F Ziegler
- Benaroya Research Institute at Virginia Mason, Seattle, Washington 98101
| | - Baohua Zhou
- From the Wells Center for Pediatric Research and Department of Pediatrics and .,Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana 46202 and
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ECD promotes gastric cancer metastasis by blocking E3 ligase ZFP91-mediated hnRNP F ubiquitination and degradation. Cell Death Dis 2018; 9:479. [PMID: 29706618 PMCID: PMC5924763 DOI: 10.1038/s41419-018-0525-x] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2018] [Accepted: 03/23/2018] [Indexed: 12/12/2022]
Abstract
The human ortholog of the Drosophila ecdysoneless gene (ECD) is required for embryonic development and cell-cycle progression; however, its role in cancer progression and metastasis remains unclear. Here, we found that ECD is frequently overexpressed in gastric cancer (GC), especially in metastatic GC, and is correlated with poor clinical outcomes in GC patients. Silencing ECD inhibited GC migration and invasion in vitro and metastasis in vivo, while ECD overexpression promoted GC migration and invasion. ECD promoted GC invasion and metastasis by protecting hnRNP F from ubiquitination and degradation. We identified ZFP91 as the E3 ubiquitin ligase that is responsible for hnRNP F ubiquitination at Lys 185 and proteasomal degradation. ECD competitively bound to hnRNP F via the N-terminal STG1 domain (13-383aa), preventing hnRNP F from interacting with ZFP91, thus preventing ZFP91-mediated hnRNP F ubiquitination and proteasomal degradation. Collectively, our findings indicate that ECD promotes cancer invasion and metastasis by preventing E3 ligase ZFP91-mediated hnRNP F ubiquitination and degradation, suggesting that ECD may be a marker for poor prognosis and a potential therapeutic target for GC patients.
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Histone demethylase JMJD1A promotes alternative splicing of AR variant 7 (AR-V7) in prostate cancer cells. Proc Natl Acad Sci U S A 2018; 115:E4584-E4593. [PMID: 29712835 DOI: 10.1073/pnas.1802415115] [Citation(s) in RCA: 72] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Formation of the androgen receptor splicing variant 7 (AR-V7) is one of the major mechanisms by which resistance of prostate cancer to androgen deprivation therapy occurs. The histone demethylase JMJD1A (Jumonji domain containing 1A) functions as a key coactivator for AR by epigenetic regulation of H3K9 methylation marks. Here, we describe a role for JMJD1A in AR-V7 expression. While JMJD1A knockdown had no effect on full-length AR (AR-FL), it reduced AR-V7 levels in prostate cancer cells. Reexpression of AR-V7 in the JMJD1A-knockdown cells elevated expression of select AR targets and partially rescued prostate cancer cell growth in vitro and in vivo. The AR-V7 protein level correlated positively with JMJD1A in a subset of human prostate cancer specimens. Mechanistically, we found that JMJD1A promoted alternative splicing of AR-V7 through heterogeneous nuclear ribonucleoprotein F (HNRNPF), a splicing factor known to regulate exon inclusion. Knockdown of JMJD1A or HNRNPF inhibited splicing of AR-V7, but not AR-FL, in a minigene reporter assay. JMJD1A was found to interact with and promote the recruitment of HNRNPF to a cryptic exon 3b on AR pre-mRNA for the generation of AR-V7. Taken together, the role of JMJD1A in AR-FL coactivation and AR-V7 alternative splicing highlights JMJD1A as a potentially promising target for prostate cancer therapy.
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Antonopoulou E, Ladomery M. Targeting Splicing in Prostate Cancer. Int J Mol Sci 2018; 19:ijms19051287. [PMID: 29693622 PMCID: PMC5983716 DOI: 10.3390/ijms19051287] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2018] [Revised: 04/18/2018] [Accepted: 04/23/2018] [Indexed: 12/22/2022] Open
Abstract
Over 95% of human genes are alternatively spliced, expressing splice isoforms that often exhibit antagonistic functions. We describe genes whose alternative splicing has been linked to prostate cancer; namely VEGFA, KLF6, BCL2L2, ERG, and AR. We discuss opportunities to develop novel therapies that target specific splice isoforms, or that target the machinery of splicing. Therapeutic approaches include the development of small molecule inhibitors of splice factor kinases, splice isoform specific siRNAs, and splice switching oligonucleotides.
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Affiliation(s)
- Effrosyni Antonopoulou
- Faculty of Health and Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK.
| | - Michael Ladomery
- Faculty of Health and Applied Sciences, University of the West of England, Coldharbour Lane, Bristol BS16 1QY, UK.
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Toledo-Leyva A, Villegas-Pineda JC, Encarnación-Guevara S, Gallardo-Rincón D, Talamás-Rohana P. Effect of ovarian cancer ascites on SKOV-3 cells proteome: new proteins associated with aggressive phenotype in epithelial ovarian cancer. Proteome Sci 2018; 16:3. [PMID: 29456457 PMCID: PMC5810116 DOI: 10.1186/s12953-018-0133-9] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2017] [Accepted: 02/06/2018] [Indexed: 12/27/2022] Open
Abstract
Background Epithelial ovarian cancer is the second most lethal gynecological cancer worldwide. Ascites can be found in all clinical stages, however in advanced disease stages IIIC and IV it is more frequent and could be massive, associated with worse prognosis. Due to the above, it was our interest to understanding how the ascites of ovarian cancer patients induces the mechanisms by which the cells present in it acquire a more aggressive phenotype and to know new proteins associated to this process. Methods A proteomic analysis of SKOV-3 cells treated with five different EOC ascites was performed by two-dimensional electrophoresis coupled to MALDI-TOF. The level of expression of the proteins of interest was validated by RT-PCR because several of these proteins have only been reported at the messenger level. Results Among the proteins identified that increased their expression in ascites-treated SKOV-3 cells, were Ran GTPase, ZNF268, and Synaptotagmin like-3. On the other hand, proteins that were negatively regulated by ascites were HLA-I, HSPB1, ARF1, Synaptotagmin 1, and hnRNPH1, among others. Furthermore, an interactome for every one of these proteins was done in order to identify biological processes, molecular actions, and cellular components in which they may participate. Conclusions Identified proteins participate in cellular processes highly relevant to the aggressive phenotype such as nuclear transport, regulation of gene expression, vesicular trafficking, evasion of the immune response, invasion, metastasis, and in resistance to chemotherapy. These proteins may represent a source of information which has the potential to be evaluated for the design of therapies directed against these malignant cells that reside on ovarian cancer ascites.
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Affiliation(s)
- Alfredo Toledo-Leyva
- 1Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, Delegación Gustavo A. Madero, 07360 Ciudad de México, Mexico.,Present address: Centro de Investigación de Cáncer en Sonora, Ciudad Obregón, Sonora 85010 Mexico
| | - Julio César Villegas-Pineda
- 1Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, Delegación Gustavo A. Madero, 07360 Ciudad de México, Mexico.,Present address: Centro de Investigación de Cáncer en Sonora, Ciudad Obregón, Sonora 85010 Mexico
| | - Sergio Encarnación-Guevara
- 2Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Av. Universidad s/n Col. Chamilpa, 62210 Cuernavaca, Morelos Mexico
| | - Dolores Gallardo-Rincón
- 3Instituto Nacional de Cancerología, Av. San Fernando No. 22, Col. Sección XVI Delegación Tlalpan, 14080 Ciudad de México, Mexico
| | - Patricia Talamás-Rohana
- 1Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, Delegación Gustavo A. Madero, 07360 Ciudad de México, Mexico
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hnRNP A1/A2 and Sam68 collaborate with SRSF10 to control the alternative splicing response to oxaliplatin-mediated DNA damage. Sci Rep 2018; 8:2206. [PMID: 29396485 PMCID: PMC5797138 DOI: 10.1038/s41598-018-20360-x] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2017] [Accepted: 01/17/2018] [Indexed: 12/02/2022] Open
Abstract
Little is known about how RNA binding proteins cooperate to control splicing, and how stress pathways reconfigure these assemblies to alter splice site selection. We have shown previously that SRSF10 plays an important role in the Bcl-x splicing response to DNA damage elicited by oxaliplatin in 293 cells. Here, RNA affinity assays using a portion of the Bcl-x transcript required for this response led to the recovery of the SRSF10-interacting protein 14-3-3ε and the Sam68-interacting protein hnRNP A1. Although SRSF10, 14-3-3ε, hnRNP A1/A2 and Sam68 do not make major contributions to the regulation of Bcl-x splicing under normal growth conditions, upon DNA damage they become important to activate the 5′ splice site of pro-apoptotic Bcl-xS. Our results indicate that DNA damage reconfigures the binding and activity of several regulatory RNA binding proteins on the Bcl-x pre-mRNA. Moreover, SRSF10, hnRNP A1/A2 and Sam68 collaborate to drive the DNA damage-induced splicing response of several transcripts that produce components implicated in apoptosis, cell-cycle control and DNA repair. Our study reveals how the circuitry of splicing factors is rewired to produce partnerships that coordinate alternative splicing across processes crucial for cell fate.
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Li Y, Bakke J, Finkelstein D, Zeng H, Wu J, Chen T. HNRNPH1 is required for rhabdomyosarcoma cell growth and survival. Oncogenesis 2018; 7:9. [PMID: 29362363 PMCID: PMC5833419 DOI: 10.1038/s41389-017-0024-4] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2017] [Accepted: 12/12/2017] [Indexed: 11/21/2022] Open
Abstract
Rhabdomyosarcoma (RMS) is an aggressive and difficult to treat cancer characterized by a muscle-like phenotype. Although the average 5-y survival rate is 65% for newly diagnosed RMS, the treatment options for metastatic disease are limited in efficacy, with the 5-y survival rate plummeting to 30%. Heterogenous nuclear ribonucleoprotein H1 (HNRNPH1) is an RNA-binding protein that is highly expressed in many cancers, including RMS. To determine the role HNRNPH1 plays in RMS tumorigenesis, we investigated its expression and effect on growth in three cellular models of RMS: RD, RH30, and RH41 cells. Upon knockdown of HNRNPH1, growth of all cell lines was reduced, most likely through a combination of apoptosis and cell cycle arrest. We then recapitulated this finding by performing in vivo xenograft studies, in which knockdown of HNRNPH1 resulted in a reduction of tumor formation and growth. We used RNA sequencing to identify changes in gene expression after HNRNPH1 knockdown and found altered splicing of some oncogenes. Our data contribute to understanding the role of HNRNPH1 in RMS development.
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Affiliation(s)
- Yanfeng Li
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Jesse Bakke
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - David Finkelstein
- Department of Computational Biology, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Hu Zeng
- Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN, USA
- Division of Rheumatology, Department of Medicine, Mayo Clinic, Rochester, MN, USA
| | - Jing Wu
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN, USA
| | - Taosheng Chen
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, TN, USA.
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Post-Transcriptional Regulation of Anti-Apoptotic BCL2 Family Members. Int J Mol Sci 2018; 19:ijms19010308. [PMID: 29361709 PMCID: PMC5796252 DOI: 10.3390/ijms19010308] [Citation(s) in RCA: 100] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2017] [Revised: 01/05/2018] [Accepted: 01/16/2018] [Indexed: 12/20/2022] Open
Abstract
Anti-apoptotic B cell lymphoma 2 (BCL2) family members (BCL2, MCL1, BCLxL, BCLW, and BFL1) are key players in the regulation of intrinsic apoptosis. Dysregulation of these proteins not only impairs normal development, but also contributes to tumor progression and resistance to various anti-cancer therapies. Therefore, cells maintain strict control over the expression of anti-apoptotic BCL2 family members using multiple mechanisms. Over the past two decades, the importance of post-transcriptional regulation of mRNA in controlling gene expression and its impact on normal homeostasis and disease have begun to be appreciated. In this review, we discuss the RNA binding proteins (RBPs) and microRNAs (miRNAs) that mediate post-transcriptional regulation of the anti-apoptotic BCL2 family members. We describe their roles and impact on alternative splicing, mRNA turnover, and mRNA subcellular localization. We also point out the importance of future studies in characterizing the crosstalk between RBPs and miRNAs in regulating anti-apoptotic BCL2 family member expression and ultimately apoptosis.
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45
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Huang H, Zhang J, Harvey SE, Hu X, Cheng C. RNA G-quadruplex secondary structure promotes alternative splicing via the RNA-binding protein hnRNPF. Genes Dev 2017; 31:2296-2309. [PMID: 29269483 PMCID: PMC5769772 DOI: 10.1101/gad.305862.117] [Citation(s) in RCA: 129] [Impact Index Per Article: 16.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2017] [Accepted: 11/22/2017] [Indexed: 12/16/2022]
Abstract
Here, Huang et al. investigated the role of RNA secondary structure in splicing regulation and show that RNA elements with G-quadruplex-forming capacity promote exon inclusion. Analysis of RNA-binding protein footprints revealed that G quadruplexes are enriched in hnRNPF-binding sites and near hnRNPF-regulated alternatively spliced exons in the human transcriptome, thus providing new insights into the regulation of alternative splicing. It is generally thought that splicing factors regulate alternative splicing through binding to RNA consensus sequences. In addition to these linear motifs, RNA secondary structure is emerging as an important layer in splicing regulation. Here we demonstrate that RNA elements with G-quadruplex-forming capacity promote exon inclusion. Destroying G-quadruplex-forming capacity while keeping G tracts intact abrogates exon inclusion. Analysis of RNA-binding protein footprints revealed that G quadruplexes are enriched in heterogeneous nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated alternatively spliced exons in the human transcriptome. Moreover, hnRNPF regulates an epithelial–mesenchymal transition (EMT)-associated CD44 isoform switch in a G-quadruplex-dependent manner, which results in inhibition of EMT. Mining breast cancer TCGA (The Cancer Genome Atlas) data sets, we demonstrate that hnRNPF negatively correlates with an EMT gene signature and positively correlates with patient survival. These data suggest a critical role for RNA G quadruplexes in regulating alternative splicing. Modulation of G-quadruplex structural integrity may control cellular processes important for tumor progression.
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Affiliation(s)
- Huilin Huang
- Division of Hematology and Oncology, Department of Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA
| | - Jing Zhang
- Division of Hematology and Oncology, Department of Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.,Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, Texas 77030, USA.,Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.,Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
| | - Samuel E Harvey
- Division of Hematology and Oncology, Department of Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.,Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, Texas 77030, USA.,Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.,Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
| | - Xiaohui Hu
- Division of Hematology and Oncology, Department of Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.,Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, Texas 77030, USA.,Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.,Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
| | - Chonghui Cheng
- Division of Hematology and Oncology, Department of Medicine, Robert H. Lurie Comprehensive Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA.,Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, Texas 77030, USA.,Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.,Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030, USA
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Trümbach D, Pfeiffer S, Poppe M, Scherb H, Doll S, Wurst W, Schick JA. ENCoRE: an efficient software for CRISPR screens identifies new players in extrinsic apoptosis. BMC Genomics 2017; 18:905. [PMID: 29178829 PMCID: PMC5702081 DOI: 10.1186/s12864-017-4285-2] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2017] [Accepted: 11/07/2017] [Indexed: 12/13/2022] Open
Abstract
BACKGROUND As CRISPR/Cas9 mediated screens with pooled guide libraries in somatic cells become increasingly established, an unmet need for rapid and accurate companion informatics tools has emerged. We have developed a lightweight and efficient software to easily manipulate large raw next generation sequencing datasets derived from such screens into informative relational context with graphical support. The advantages of the software entitled ENCoRE (Easy NGS-to-Gene CRISPR REsults) include a simple graphical workflow, platform independence, local and fast multithreaded processing, data pre-processing and gene mapping with custom library import. RESULTS We demonstrate the capabilities of ENCoRE to interrogate results from a pooled CRISPR cellular viability screen following Tumor Necrosis Factor-alpha challenge. The results not only identified stereotypical players in extrinsic apoptotic signaling but two as yet uncharacterized members of the extrinsic apoptotic cascade, Smg7 and Ces2a. We further validated and characterized cell lines containing mutations in these genes against a panel of cell death stimuli and involvement in p53 signaling. CONCLUSIONS In summary, this software enables bench scientists with sensitive data or without access to informatic cores to rapidly interpret results from large scale experiments resulting from pooled CRISPR/Cas9 library screens.
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Affiliation(s)
- Dietrich Trümbach
- Institute of Developmental Genetics, Helmholtz Zentrum Munich, Ingolstädter Landstraße 1, 85764, Neuherberg, Germany
| | - Susanne Pfeiffer
- Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum Munich, Ingolstädter Landstraße 1, 85764, Neuherberg, Germany
| | - Manuel Poppe
- Institute of Developmental Genetics, Helmholtz Zentrum Munich, Ingolstädter Landstraße 1, 85764, Neuherberg, Germany
| | - Hagen Scherb
- Institute of Computational Biology, Helmholtz Zentrum Munich, Ingolstädter Landstraße 1, 85764, Neuherberg, Germany
| | - Sebastian Doll
- Institute of Developmental Genetics, Helmholtz Zentrum Munich, Ingolstädter Landstraße 1, 85764, Neuherberg, Germany
| | - Wolfgang Wurst
- Institute of Developmental Genetics, Helmholtz Zentrum Munich, Ingolstädter Landstraße 1, 85764, Neuherberg, Germany.,Technische Universität München-Weihenstephan, Chair of Developmental Genetics c/o Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764, Neuherberg/Munich, Germany.,German Center for Neurodegenerative Diseases (DZNE) Site Munich, Feodor-Lynen-Str. 17, 81377, Munich, Germany.,Munich Cluster for Systems Neurology (SyNergy), Feodor-Lynen-Str. 17, 81377, Munich, Germany
| | - Joel A Schick
- Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum Munich, Ingolstädter Landstraße 1, 85764, Neuherberg, Germany.
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Shkreta L, Toutant J, Durand M, Manley JL, Chabot B. SRSF10 Connects DNA Damage to the Alternative Splicing of Transcripts Encoding Apoptosis, Cell-Cycle Control, and DNA Repair Factors. Cell Rep 2017; 17:1990-2003. [PMID: 27851963 PMCID: PMC5483951 DOI: 10.1016/j.celrep.2016.10.071] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2015] [Revised: 04/19/2016] [Accepted: 10/20/2016] [Indexed: 11/12/2022] Open
Abstract
RNA binding proteins and signaling components control the production of pro-death and pro-survival splice variants of Bcl-x. DNA damage promoted by oxaliplatin increases the level of pro-apoptotic Bcl-xS in an ATM/CHK2-dependent manner, but how this shift is enforced is not known. Here, we show that in normally growing cells, when the 5′ splice site of Bcl-xS is largely repressed, SRSF10 partially relieves repression and interacts with repressor hnRNP K and stimulatory hnRNP F/H proteins. Oxaliplatin abrogates the interaction of SRSF10 with hnRNP F/H and decreases the association of SRSF10 and hnRNP K with the Bcl-x pre-mRNA. Dephosphorylation of SRSF10 is linked with these changes. A broader analysis reveals that DNA damage co-opts SRSF10 to control splicing decisions in transcripts encoding components involved in DNA repair, cell-cycle control, and apoptosis. DNA damage therefore alters the interactions between splicing regulators to elicit a splicing response that determines cell fate.
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Affiliation(s)
- Lulzim Shkreta
- Département de Microbiologie et d'Infectiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada
| | - Johanne Toutant
- Département de Microbiologie et d'Infectiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada
| | - Mathieu Durand
- Laboratory of Functional Genomics, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada
| | - James L Manley
- Department of Biological Sciences, Columbia University, New York, NY, 10027, USA
| | - Benoit Chabot
- Département de Microbiologie et d'Infectiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC J1E 4K8, Canada.
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Baralle M, Baralle FE. The splicing code. Biosystems 2017; 164:39-48. [PMID: 29122587 DOI: 10.1016/j.biosystems.2017.11.002] [Citation(s) in RCA: 44] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2017] [Revised: 11/02/2017] [Accepted: 11/03/2017] [Indexed: 01/09/2023]
Abstract
This issue dedicated to the code of life tackles very challenging and open questions in Biology. The genetic code, brilliantly uncovered over 50 years ago is an example of a univocal biological code. In fact, except for very few and marginal variations, it is the same from bacteria to man, the RNA stretch: 5' GUGUUC 3' reads as the dipeptide: Val-Phe in bacteria, in yeast, in Arabidopsis, in zebra fish, in mouse and in human. A degree of ambiguity is possible if mutations are introduced in the tRNAs in a way that the anticodon reads one amino acid but the aminoacyl-transferase attaches a different one onto the tRNA. These were the very useful suppressor genes that aided greatly the study of bacterial genetics. Other biological codes however, are more akin to social codes and are less amenable to an unambiguous deciphering. Legal and ethical codes, weather we like it or not, are flexible and depend on the structure and history of the society that has produced them, as well as a specific point in time. The codes that govern RNA splicing have similar characteristics. In fact, the splicing code depends on a myriad of different factors that in part are influenced by the background in which they are read such as different cells, tissues or developmental stages. Given the complexity of the splicing process, the construction of an algorithm that can define exons or their fate with certainty has not yet been achieved. However a substantial amount of information towards the deciphering of the splicing code has been gathered and in this manuscript we summarize the point reached.
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Affiliation(s)
- Marco Baralle
- International Centre for Genetic Engineering and Biotechnology, Padriciano 99, Italy.
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Nazim M, Masuda A, Rahman MA, Nasrin F, Takeda JI, Ohe K, Ohkawara B, Ito M, Ohno K. Competitive regulation of alternative splicing and alternative polyadenylation by hnRNP H and CstF64 determines acetylcholinesterase isoforms. Nucleic Acids Res 2017; 45:1455-1468. [PMID: 28180311 PMCID: PMC5388418 DOI: 10.1093/nar/gkw823] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/06/2016] [Revised: 08/11/2016] [Accepted: 09/07/2016] [Indexed: 12/21/2022] Open
Abstract
Acetylcholinesterase (AChE), encoded by the ACHE gene, hydrolyzes the neurotransmitter acetylcholine to terminate synaptic transmission. Alternative splicing close to the 3΄ end generates three distinct isoforms of AChET, AChEH and AChER. We found that hnRNP H binds to two specific G-runs in exon 5a of human ACHE and activates the distal alternative 3΄ splice site (ss) between exons 5a and 5b to generate AChET. Specific effect of hnRNP H was corroborated by siRNA-mediated knockdown and artificial tethering of hnRNP H. Furthermore, hnRNP H competes for binding of CstF64 to the overlapping binding sites in exon 5a, and suppresses the selection of a cryptic polyadenylation site (PAS), which additionally ensures transcription of the distal 3΄ ss required for the generation of AChET. Expression levels of hnRNP H were positively correlated with the proportions of the AChET isoform in three different cell lines. HnRNP H thus critically generates AChET by enhancing the distal 3΄ ss and by suppressing the cryptic PAS. Global analysis of CLIP-seq and RNA-seq also revealed that hnRNP H competitively regulates alternative 3΄ ss and alternative PAS in other genes. We propose that hnRNP H is an essential factor that competitively regulates alternative splicing and alternative polyadenylation.
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Affiliation(s)
- Mohammad Nazim
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Akio Masuda
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Mohammad Alinoor Rahman
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Farhana Nasrin
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Jun-Ichi Takeda
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Kenji Ohe
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Bisei Ohkawara
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Mikako Ito
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
| | - Kinji Ohno
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan
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Molecular insights into the specific recognition between the RNA binding domain qRRM2 of hnRNP F and G-tract RNA: A molecular dynamics study. Biochem Biophys Res Commun 2017; 494:95-100. [PMID: 29050934 DOI: 10.1016/j.bbrc.2017.10.078] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2017] [Accepted: 10/15/2017] [Indexed: 01/21/2023]
Abstract
Heterogeneous nuclear ribonucleoprotein F (hnRNP F) controls the expression of various genes through regulating the alternative splicing of pre-mRNAs in the nucleus. It uses three quasi-RNA recognition motifs (qRRMs) to recognize G-tract RNA which contains at least three consecutive guanines. The structures containing qRRMs of hnRNP F in complex with G-tract RNA have been determined by nuclear magnetic resonance (NMR) spectroscopy, shedding light on the recognition mechanism of qRRMs with G-tract RNA. However, knowledge of the recognition details is still lacking. To investigate how qRRMs specifically bind with G-tract RNA and how the mutations of any guanine to an adenine in the G-tract affect the binding, molecular dynamics simulations with binding free energy analysis were performed based on the NMR structure of qRRM2 in complex with G-tract RNA. Simulation results demonstrate that qRRM2 binds strongly with G-tract RNA, but any mutation of the G-tract leads to a drastic reduction of the binding free energy. Further comparisons of the energetic components reveal that van der Waals and non-polar interactions play essential roles in the binding between qRRM2 and G-tract RNA, but the interactions are weakened by the effect of RNA mutations. Structural and dynamical analyses indicate that when qRRM2 binds with G-tract RNA, both qRRM2 and G-tract maintain stabilized structures and dynamics; however, the stability is disrupted by the mutations of the G-tract. These results provide novel insights into the recognition mechanism of qRRM2 with G-tract RNA that are not elucidated by the NMR technique.
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