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1
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Nasrin F, Nagar P, Islam M, Heeamoni S, Hasan M, Ohno K, Rahman M. SRSF6 and SRSF1 coordinately enhance the inclusion of human MUSK exon 10 to generate a Wnt-sensitive MuSK isoform. NAR MOLECULAR MEDICINE 2025; 2:ugaf007. [PMID: 40161265 PMCID: PMC11954543 DOI: 10.1093/narmme/ugaf007] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/02/2025] [Revised: 03/07/2025] [Accepted: 03/18/2025] [Indexed: 04/02/2025]
Abstract
Alternative splicing in genes associated with neuromuscular junction (NMJ) often compromises neuromuscular signal transmission and provokes pathological consequences. Muscle-specific receptor tyrosine kinase (MuSK) is an essential molecule in the NMJ. MUSK exon 10 encodes an important part of the frizzled-like cysteine-rich domain, which is necessary for Wnt-mediated acetylcholine receptors clustering at NMJ. MUSK exon 10 is alternatively spliced in humans but not in mice. We reported that humans acquired a unique exonic splicing silencer in exon 10 compared to mice, which promotes exon skipping coordinated by hnRNP C, YB-1, and hnRNP L. Here, we have dissected the underlying mechanisms of exon inclusion. We precisely characterized the exonic splicing enhancer (ESE) elements and determined the functional motifs. We demonstrated that SRSF6 and SRSF1 coordinately enhance exon inclusion through multiple functional motifs in the ESE. Remarkably, SRSF6 exerts a stronger effect than SRSF1, and SRSF6 alone can compensate the function of SRSF1. Interestingly, differentiated muscle reduces the expression of splicing suppressors, rather than enhancers, to generate a functional Wnt-sensitive MuSK isoform to promote neuromuscular signal transmission. Finally, we developed splice-switching antisense oligonucleotides, which could be used to selectively modulate the expression of MUSK isoforms toward a beneficial outcome for therapeutic intervention.
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Affiliation(s)
- Farhana Nasrin
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, 4668550 Aichi, Japan
| | - Preeti Nagar
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States
| | - Md Rafikul Islam
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States
| | - Shabiha Afroj Heeamoni
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States
| | - Md Mahbub Hasan
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States
| | - Kinji Ohno
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, 4668550 Aichi, Japan
- Graduate School of Nutritional Sciences, Nagoya University of Arts and Sciences, Nisshin, 4700196 Aichi, Japan
| | - Mohammad Alinoor Rahman
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, United States
- Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
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2
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Jia Z, Wang J, Meng X, Yang X, Tian Y, Wang B, Chen M, Yang J, Das D, Cao Y. Evolution and stress response potential of the plant splicing factor U1C. Sci Rep 2024; 14:17212. [PMID: 39060315 PMCID: PMC11282270 DOI: 10.1038/s41598-024-68190-4] [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: 06/27/2023] [Accepted: 07/22/2024] [Indexed: 07/28/2024] Open
Abstract
Alternative splicing is a crucial process in multicellular eukaryote, facilitated by the assembly of spliceosomal complexes comprising numerous small ribonucleoproteins. At an early stage, U1C is thought to be required for 5' splice site recognition and base pairing. However, a systematic analysis of the U1C gene family in response to developmental cues and stress conditions has not yet been conducted in plants. This study identified 114 U1C genes in 72 plant species using basic bioinformatics analyses. Phylogenetic analysis was used to compare gene and protein structures, promoter motifs, and tissue- and stress-specific expression levels, revealing their functional commonalities or diversity in response to developmental cues, such as embryonic expression, or stress treatments, including drought and heat. Fluorescence quantitative expression analysis showed that U1C gene expression changed under salt, low temperature, drought, and Cd stress in rice seedlings. However, gene expression in shoots and roots was not consistent under different stress conditions, suggesting a complex regulatory mechanism. This research provides foundational insights into the U1C gene family's role in plant development and stress responses, highlighting potential targets for future studies.
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Affiliation(s)
- Zichang Jia
- School of Life Sciences, Nantong University, 9 Seyuan Road, Nantong, 226019, Jiangsu, China
- State Key Laboratory of Crop Biology, College of Life Science, Shandong Agricultural University, Taian, China
| | - Junjie Wang
- School of Life Sciences, Nantong University, 9 Seyuan Road, Nantong, 226019, Jiangsu, China
- Co-Innovation Center for Sustainable Forestry in Southern China and Key Laboratory of National Forestry and Grassland Administration on Subtropical Forest Biodiversity Conservation, College of Biology and the Environment, Nanjing Forestry University, Nanjing, China
| | - Xiangfeng Meng
- School of Life Sciences, Nantong University, 9 Seyuan Road, Nantong, 226019, Jiangsu, China
| | - Xue Yang
- State Key Laboratory of Crop Biology, College of Life Science, Shandong Agricultural University, Taian, China
| | - Yuan Tian
- State Key Laboratory of Crop Biology, College of Life Science, Shandong Agricultural University, Taian, China
| | - Baohua Wang
- School of Life Sciences, Nantong University, 9 Seyuan Road, Nantong, 226019, Jiangsu, China
| | - Moxian Chen
- Co-Innovation Center for Sustainable Forestry in Southern China and Key Laboratory of National Forestry and Grassland Administration on Subtropical Forest Biodiversity Conservation, College of Biology and the Environment, Nanjing Forestry University, Nanjing, China
| | - Jingfang Yang
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China
| | - Debatosh Das
- College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, MO, 65201, USA.
| | - Yunying Cao
- School of Life Sciences, Nantong University, 9 Seyuan Road, Nantong, 226019, Jiangsu, China.
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Han Y, Han J, Li Z, Chen S, Liu J, Zhou R, Zhao S, Li D, Liu Z, Zhao Y, Hao J, Chai G. Identification and characterization of a novel intronic splicing mutation in CSF1R-related leukoencephalopathy. CNS Neurosci Ther 2024; 30:e14815. [PMID: 38922778 PMCID: PMC11194178 DOI: 10.1111/cns.14815] [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: 10/25/2023] [Revised: 04/16/2024] [Accepted: 05/08/2024] [Indexed: 06/28/2024] Open
Abstract
AIMS Colony stimulating factor 1 receptor (CSF1R)-related leukoencephalopathy is a rapidly progressing neurodegenerative disease caused by CSF1R gene mutations. This study aimed to identify and investigate the effect of a novel intronic mutation (c.1754-3C>G) of CSF1R on splicing. METHODS A novel intronic mutation was identified using whole-exome sequencing. To investigate the impact of this mutation, we employed various bioinformatics tools to analyze the transcription of the CSF1R gene and the three-dimensional structure of its encoded protein. Furthermore, reverse transcription polymerase chain reaction (RT-PCR) was performed to validate the findings. RESULTS A novel mutation (c.1754-3C>G) in CSF1R was identified, which results in exon 13 skipping due to the disruption of the 3' splice site consensus sequence NYAG/G. This exon skipping event was further validated in the peripheral blood of the mutation carrier through RT-PCR and Sanger sequencing. Protein structure prediction indicated a disruption in the tyrosine kinase domain, with the truncated protein showing significant structural alterations. CONCLUSIONS Our findings underscore the importance of intronic mis-splicing mutations in the diagnosis and management of CSF1R-related leukoencephalopathy.
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Affiliation(s)
- Yilai Han
- Department of NeurologyXuanwu Hospital Capital Medical University, National Center for Neurological DisordersBeijingChina
| | - Jinming Han
- Department of NeurologyXuanwu Hospital Capital Medical University, National Center for Neurological DisordersBeijingChina
| | - Zhen Li
- Department of NeurologyXuanwu Hospital Capital Medical University, National Center for Neurological DisordersBeijingChina
| | - Siqi Chen
- Department of NeurologyXuanwu Hospital Capital Medical University, National Center for Neurological DisordersBeijingChina
| | - Ju Liu
- Department of NeurologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouHenanChina
| | - Ruxing Zhou
- Department of NeurologyXuanwu Hospital Capital Medical University, National Center for Neurological DisordersBeijingChina
| | - Shufang Zhao
- Department of NeurologyXuanwu Hospital Capital Medical University, National Center for Neurological DisordersBeijingChina
| | - Dawei Li
- Department of NeurologyXuanwu Hospital Capital Medical University, National Center for Neurological DisordersBeijingChina
| | - Zheng Liu
- Department of NeurologyXuanwu Hospital Capital Medical University, National Center for Neurological DisordersBeijingChina
| | - Yinan Zhao
- Department of NeurologyXuanwu Hospital Capital Medical University, National Center for Neurological DisordersBeijingChina
| | - Junwei Hao
- Department of NeurologyXuanwu Hospital Capital Medical University, National Center for Neurological DisordersBeijingChina
- Beijing Municipal Geriatric Medical Research CenterBeijingChina
- Key Laboratory for Neurodegenerative Diseases of Ministry of EducationBeijingChina
| | - Guoliang Chai
- Department of NeurologyXuanwu Hospital Capital Medical University, National Center for Neurological DisordersBeijingChina
- Beijing Municipal Geriatric Medical Research CenterBeijingChina
- Chinese Institutes for Medical ResearchBeijingChina
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4
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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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5
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Rodriguez Gallo MC, Uhrig RG. Phosphorylation mediated regulation of RNA splicing in plants. FRONTIERS IN PLANT SCIENCE 2023; 14:1249057. [PMID: 37780493 PMCID: PMC10539000 DOI: 10.3389/fpls.2023.1249057] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 06/28/2023] [Accepted: 08/22/2023] [Indexed: 10/03/2023]
Abstract
For the past two decades, the study of alternative splicing (AS) and its involvement in plant development and stress response has grown in popularity. Only recently however, has the focus shifted to the study of how AS regulation (or lack-thereof) affects downstream mRNA and protein landscapes and how these AS regulatory events impact plant development and stress tolerance. In humans, protein phosphorylation represents one of the predominant mechanisms by which AS is regulated and thus the protein kinases governing these phosphorylation events are of interest for further study. Large-scale phosphoproteomic studies in plants have consistently found that RNA splicing-related proteins are extensively phosphorylated, however, the signaling pathways involved in AS regulation have not been resolved. In this mini-review, we summarize our current knowledge of the three major splicing-related protein kinase families in plants that are suggested to mediate AS phospho-regulation and draw comparisons to their metazoan orthologs. We also summarize and contextualize the phosphorylation events identified as occurring on splicing-related protein families to illustrate the high degree to which splicing-related proteins are modified, placing a new focus on elucidating the impacts of AS at the protein and PTM-level.
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Affiliation(s)
| | - R. Glen Uhrig
- University of Alberta, Department of Biological Sciences, Edmonton, AB, Canada
- University of Alberta, Department of Biochemistry, Edmonton, AB, Canada
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6
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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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7
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Lim WF, Rinaldi C. RNA Transcript Diversity in Neuromuscular Research. J Neuromuscul Dis 2023:JND221601. [PMID: 37182892 DOI: 10.3233/jnd-221601] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/16/2023]
Abstract
Three decades since the Human Genome Project began, scientists have now identified more then 25,000 protein coding genes in the human genome. The vast majority of the protein coding genes (> 90%) are multi-exonic, with the coding DNA being interrupted by intronic sequences, which are removed from the pre-mRNA transcripts before being translated into proteins, a process called splicing maturation. Variations in this process, i.e. by exon skipping, intron retention, alternative 5' splice site (5'ss), 3' splice site (3'ss), or polyadenylation usage, lead to remarkable transcriptome and proteome diversity in human tissues. Given its critical biological importance, alternative splicing is tightly regulated in a tissue- and developmental stage-specific manner. The central nervous system and skeletal muscle are amongst the tissues with the highest number of differentially expressed alternative exons, revealing a remarkable degree of transcriptome complexity. It is therefore not surprising that splicing mis-regulation is causally associated with a myriad of neuromuscular diseases, including but not limited to amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), Duchenne muscular dystrophy (DMD), and myotonic dystrophy type 1 and 2 (DM1, DM2). A gene's transcript diversity has since become an integral and an important consideration for drug design, development and therapy. In this review, we will discuss transcript diversity in the context of neuromuscular diseases and current approaches to address splicing mis-regulation.
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Affiliation(s)
- Wooi Fang Lim
- Department of Paediatrics and Institute of Developmental and Regenerative Medicine, University of Oxford, Oxford, UK
| | - Carlo Rinaldi
- Department of Paediatrics and Institute of Developmental and Regenerative Medicine, University of Oxford, Oxford, UK
- MDUK Oxford Neuromuscular Centre, University of Oxford, Oxford, UK
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8
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Bushra S, Lin YN, Joudaki A, Ito M, Ohkawara B, Ohno K, Masuda A. Neural Isoforms of Agrin Are Generated by Reduced PTBP1-RNA Interaction Network Spanning the Neuron-Specific Splicing Regions in AGRN. Int J Mol Sci 2023; 24:ijms24087420. [PMID: 37108583 PMCID: PMC10139058 DOI: 10.3390/ijms24087420] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2023] [Revised: 04/12/2023] [Accepted: 04/14/2023] [Indexed: 04/29/2023] Open
Abstract
Agrin is a heparan sulfate proteoglycan essential for the clustering of acetylcholine receptors at the neuromuscular junction. Neuron-specific isoforms of agrin are generated by alternative inclusion of three exons, called Y, Z8, and Z11 exons, although their processing mechanisms remain elusive. We found, by inspection of splicing cis-elements into the human AGRN gene, that binding sites for polypyrimidine tract binding protein 1 (PTBP1) were extensively enriched around Y and Z exons. PTBP1-silencing enhanced the coordinated inclusion of Y and Z exons in human SH-SY5Y neuronal cells, even though three constitutive exons are flanked by these alternative exons. Deletion analysis using minigenes identified five PTBP1-binding sites with remarkable splicing repression activities around Y and Z exons. Furthermore, artificial tethering experiments indicated that binding of a single PTBP1 molecule to any of these sites represses nearby Y or Z exons as well as the other distal exons. The RRM4 domain of PTBP1, which is required for looping out a target RNA segment, was likely to play a crucial role in the repression. Neuronal differentiation downregulates PTBP1 expression and promotes the coordinated inclusion of Y and Z exons. We propose that the reduction in the PTPB1-RNA network spanning these alternative exons is essential for the generation of the neuron-specific agrin isoforms.
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Affiliation(s)
- Samira Bushra
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Aichi, Japan
| | - Ying-Ni Lin
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Aichi, Japan
| | - Atefeh Joudaki
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Aichi, Japan
| | - Mikako Ito
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Aichi, Japan
| | - Bisei Ohkawara
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Aichi, Japan
| | - Kinji Ohno
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Aichi, Japan
| | - Akio Masuda
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Aichi, Japan
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9
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Verdile V, Guizzo G, Ferrante G, Paronetto MP. RNA Targeting in Inherited Neuromuscular Disorders: Novel Therapeutic Strategies to Counteract Mis-Splicing. Cells 2021; 10:2850. [PMID: 34831073 PMCID: PMC8616048 DOI: 10.3390/cells10112850] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2021] [Revised: 10/08/2021] [Accepted: 10/18/2021] [Indexed: 01/14/2023] Open
Abstract
Neuromuscular disorders represent multifaceted abnormal conditions, with little or no cure, leading to patient deaths from complete muscle wasting and atrophy. Despite strong efforts in the past decades, development of effective treatments is still urgently needed. Advent of next-generation sequencing technologies has allowed identification of novel genes and mutations associated with neuromuscular pathologies, highlighting splicing defects as essential players. Deciphering the significance and relative contributions of defective RNA metabolism will be instrumental to address and counteract these malignancies. We review here recent progress on the role played by alternative splicing in ensuring functional neuromuscular junctions (NMJs), and its involvement in the pathogenesis of NMJ-related neuromuscular disorders, with particular emphasis on congenital myasthenic syndromes and muscular dystrophies. We will also discuss novel strategies based on oligonucleotides designed to bind their cognate sequences in the RNA or targeting intermediary of mRNA metabolism. These efforts resulted in several chemical classes of RNA molecules that have recently proven to be clinically effective, more potent and better tolerated than previous strategies.
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Affiliation(s)
- Veronica Verdile
- Laboratory of Molecular and Cellular Neurobiology, Fondazione Santa Lucia, CERC, 00143 Rome, Italy; (V.V.); (G.G.); (G.F.)
- Department of Movement, Human and Health Sciences, University of Rome Foro Italico, Piazza Lauro de Bosis 6, 00135 Rome, Italy
| | - Gloria Guizzo
- Laboratory of Molecular and Cellular Neurobiology, Fondazione Santa Lucia, CERC, 00143 Rome, Italy; (V.V.); (G.G.); (G.F.)
| | - Gabriele Ferrante
- Laboratory of Molecular and Cellular Neurobiology, Fondazione Santa Lucia, CERC, 00143 Rome, Italy; (V.V.); (G.G.); (G.F.)
| | - Maria Paola Paronetto
- Laboratory of Molecular and Cellular Neurobiology, Fondazione Santa Lucia, CERC, 00143 Rome, Italy; (V.V.); (G.G.); (G.F.)
- Department of Movement, Human and Health Sciences, University of Rome Foro Italico, Piazza Lauro de Bosis 6, 00135 Rome, Italy
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10
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Zhang KL, Zhou JL, Yang JF, Zhao YZ, Das D, Hao GF, Wu C, Zhang J, Zhu FY, Chen MX, Zhou SM. Phylogenetic Comparison and Splicing Analysis of the U1 snRNP-specific Protein U1C in Eukaryotes. Front Mol Biosci 2021; 8:696319. [PMID: 34568424 PMCID: PMC8458698 DOI: 10.3389/fmolb.2021.696319] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2021] [Accepted: 08/26/2021] [Indexed: 11/13/2022] Open
Abstract
As a pivotal regulator of 5’ splice site recognition, U1 small nuclear ribonucleoprotein (U1 snRNP)-specific protein C (U1C) regulates pre-mRNA splicing by interacting with other components of the U1 snRNP complex. Previous studies have shown that U1 snRNP and its components are linked to a variety of diseases, including cancer. However, the phylogenetic relationships and expression profiles of U1C have not been studied systematically. To this end, we identified a total of 110 animal U1C genes and compared them to homologues from yeast and plants. Bioinformatics analysis shows that the structure and function of U1C proteins is relatively conserved and is found in multiple copies in a few members of the U1C gene family. Furthermore, the expression patterns reveal that U1Cs have potential roles in cancer progression and human development. In summary, our study presents a comprehensive overview of the animal U1C gene family, which can provide fundamental data and potential cues for further research in deciphering the molecular function of this splicing regulator.
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Affiliation(s)
- Kai-Lu Zhang
- Division of Gastroenterology, Shenzhen Children's Hospital, Shenzhen, China.,Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Nanjing, China.,State Key Laboratory of Crop Biology, College of Life Science, Shandong Agricultural University, Taian, China
| | - Jian-Li Zhou
- Division of Gastroenterology, Shenzhen Children's Hospital, Shenzhen, China
| | - Jing-Fang Yang
- Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, China
| | - Yu-Zhen Zhao
- Division of Gastroenterology, Shenzhen Children's Hospital, Shenzhen, China
| | - Debatosh Das
- Department of Biology, Hong Kong Baptist University, and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong, SAR China
| | - Ge-Fei Hao
- Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, China
| | - Caie Wu
- College of Light Industry and Food Engineering, Nanjing Forestry University, Jiangsu, China
| | - Jianhua Zhang
- Department of Biology, Hong Kong Baptist University, and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong, SAR China
| | - Fu-Yuan Zhu
- Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Nanjing, China
| | - Mo-Xian Chen
- Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Nanjing, China
| | - Shao-Ming Zhou
- Division of Gastroenterology, Shenzhen Children's Hospital, Shenzhen, China
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11
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Deletion of RBMX RGG/RG motif in Shashi-XLID syndrome leads to aberrant p53 activation and neuronal differentiation defects. Cell Rep 2021; 36:109337. [PMID: 34260915 DOI: 10.1016/j.celrep.2021.109337] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2020] [Revised: 04/29/2021] [Accepted: 06/11/2021] [Indexed: 01/01/2023] Open
Abstract
RNA-binding proteins play important roles in X-linked intellectual disability (XLID). In this study, we investigate the contribution of the XLID-associated RBMX in neuronal differentiation. We show that RBMX-depleted cells exhibit aberrant activation of the p53 pathway. Moreover, we identify that the RBMX RGG/RG motif is methylated by protein arginine methyltransferase 5 (PRMT5), and this regulates assembly with the SRSF1 splicing factor into higher-order complexes. Depletion of RBMX or disruption of the RBMX/SRSF1 complex in PRMT5-depleted cells reduces SRSF1 binding to the MDM4 precursor (pre-)mRNA, leading to exon 6 exclusion and lower MDM4 protein levels. Transcriptomic analysis of isogenic Shashi-XLID human-induced pluripotent stem cells (hiPSCs) generated using CRISPR-Cas9 reveals a dysregulation of MDM4 splicing and aberrant p53 upregulation. Shashi-XLID neural progenitor cells (NPCs) display differentiation and morphological abnormalities accompanied with excessive apoptosis. Our findings identify RBMX as a regulator of SRSF1 and the p53 pathway, suggesting that the loss of function of the RBMX RGG/RG motif is the cause of Shashi-XLID syndrome.
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12
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AKSOY G, LÜLEYAP Ü, EVYAPAN G, PAZARCI P, ALPTEKİN D, PAZARBAŞI A, YILMAZ MB. Sh-Sy5y hücre hattında sodyum bütiratın bazı alternatif kırpılma genleri ve BACE1 izoformları üzeindeki etkisi. CUKUROVA MEDICAL JOURNAL 2021. [DOI: 10.17826/cumj.870361] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/07/2022] Open
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13
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Fan T, Zhao YZ, Yang JF, Liu QL, Tian Y, Debatosh D, Liu YG, Zhang J, Chen C, Chen MX, Zhou SM. Phylogenetic comparison and splice site conservation of eukaryotic U1 snRNP-specific U1-70K gene family. Sci Rep 2021; 11:12760. [PMID: 34140531 PMCID: PMC8211703 DOI: 10.1038/s41598-021-91693-3] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2020] [Accepted: 05/05/2021] [Indexed: 02/05/2023] Open
Abstract
Eukaryotic cells can expand their coding ability by using their splicing machinery, spliceosome, to process precursor mRNA (pre-mRNA) into mature messenger RNA. The mega-macromolecular spliceosome contains multiple subcomplexes, referred to as small nuclear ribonucleoproteins (snRNPs). Among these, U1 snRNP and its central component, U1-70K, are crucial for splice site recognition during early spliceosome assembly. The human U1-70K has been linked to several types of human autoimmune and neurodegenerative diseases. However, its phylogenetic relationship has been seldom reported. To this end, we carried out a systemic analysis of 95 animal U1-70K genes and compare these proteins to their yeast and plant counterparts. Analysis of their gene and protein structures, expression patterns and splicing conservation suggest that animal U1-70Ks are conserved in their molecular function, and may play essential role in cancers and juvenile development. In particular, animal U1-70Ks display unique characteristics of single copy number and a splicing isoform with truncated C-terminal, suggesting the specific role of these U1-70Ks in animal kingdom. In summary, our results provide phylogenetic overview of U1-70K gene family in vertebrates. In silico analyses conducted in this work will act as a reference for future functional studies of this crucial U1 splicing factor in animal kingdom.
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Affiliation(s)
- Tao Fan
- grid.452787.b0000 0004 1806 5224Division of Gastroenterology, Shenzhen Children’s Hospital, Shenzhen, 518038 People’s Republic of China ,grid.440622.60000 0000 9482 4676State Key Laboratory of Crop Biology, College of Life Science, Shandong Agricultural University, Taian, Shandong People’s Republic of China ,grid.10784.3a0000 0004 1937 0482Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, People’s Republic of China
| | - Yu-Zhen Zhao
- grid.452787.b0000 0004 1806 5224Division of Gastroenterology, Shenzhen Children’s Hospital, Shenzhen, 518038 People’s Republic of China
| | - Jing-Fang Yang
- grid.411407.70000 0004 1760 2614Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, 430079 People’s Republic of China
| | - Qin-Lai Liu
- School of Basic Medicine, Shandong First Medical University and Shandong Academy of Medical Sciences, Qingdao, People’s Republic of China
| | - Yuan Tian
- grid.440622.60000 0000 9482 4676State Key Laboratory of Crop Biology, College of Life Science, Shandong Agricultural University, Taian, Shandong People’s Republic of China ,grid.10784.3a0000 0004 1937 0482Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, People’s Republic of China
| | - Das Debatosh
- grid.10784.3a0000 0004 1937 0482Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, People’s Republic of China
| | - Ying-Gao Liu
- grid.440622.60000 0000 9482 4676State Key Laboratory of Crop Biology, College of Life Science, Shandong Agricultural University, Taian, Shandong People’s Republic of China
| | - Jianhua Zhang
- grid.10784.3a0000 0004 1937 0482Department of Biology, Hong Kong Baptist University, and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, Hong Kong
| | - Chen Chen
- grid.410745.30000 0004 1765 1045Department of Infectious Disease, Nanjing Infectious Disease Center, The Second Hospital of Nanjing, Nanjing University of Chinese Medicine, Nanjing, 210003 People’s Republic of China
| | - Mo-Xian Chen
- grid.452787.b0000 0004 1806 5224Division of Gastroenterology, Shenzhen Children’s Hospital, Shenzhen, 518038 People’s Republic of China
| | - Shao-Ming Zhou
- grid.452787.b0000 0004 1806 5224Division of Gastroenterology, Shenzhen Children’s Hospital, Shenzhen, 518038 People’s Republic of China
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14
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Low YH, Asi Y, Foti SC, Lashley T. Heterogeneous Nuclear Ribonucleoproteins: Implications in Neurological Diseases. Mol Neurobiol 2021; 58:631-646. [PMID: 33000450 PMCID: PMC7843550 DOI: 10.1007/s12035-020-02137-4] [Citation(s) in RCA: 41] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Accepted: 09/17/2020] [Indexed: 12/13/2022]
Abstract
Heterogenous nuclear ribonucleoproteins (hnRNPs) are a complex and functionally diverse family of RNA binding proteins with multifarious roles. They are involved, directly or indirectly, in alternative splicing, transcriptional and translational regulation, stress granule formation, cell cycle regulation, and axonal transport. It is unsurprising, given their heavy involvement in maintaining functional integrity of the cell, that their dysfunction has neurological implications. However, compared to their more established roles in cancer, the evidence of hnRNP implication in neurological diseases is still in its infancy. This review aims to consolidate the evidences for hnRNP involvement in neurological diseases, with a focus on spinal muscular atrophy (SMA), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), multiple sclerosis (MS), congenital myasthenic syndrome (CMS), and fragile X-associated tremor/ataxia syndrome (FXTAS). Understanding more about hnRNP involvement in neurological diseases can further elucidate the pathomechanisms involved in these diseases and perhaps guide future therapeutic advances.
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Affiliation(s)
- Yi-Hua Low
- The Queen Square Brain Bank for Neurological Disorders, Department of Clinical and Movement Disorders, UCL Queen Square Institute of Neurology, University College London, London, WC1N 3BG, UK
- Duke-NUS Medical School, Singapore, Singapore
| | - Yasmine Asi
- The Queen Square Brain Bank for Neurological Disorders, Department of Clinical and Movement Disorders, UCL Queen Square Institute of Neurology, University College London, London, WC1N 3BG, UK
| | - Sandrine C Foti
- The Queen Square Brain Bank for Neurological Disorders, Department of Clinical and Movement Disorders, UCL Queen Square Institute of Neurology, University College London, London, WC1N 3BG, UK
- Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, University College London, London, UK
| | - Tammaryn Lashley
- The Queen Square Brain Bank for Neurological Disorders, Department of Clinical and Movement Disorders, UCL Queen Square Institute of Neurology, University College London, London, WC1N 3BG, UK.
- Department of Neurodegenerative Disease, UCL Queen Square Institute of Neurology, University College London, London, UK.
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15
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Frederiksen SB, Holm LL, Larsen MR, Doktor TK, Andersen HS, Hastings ML, Hua Y, Krainer AR, Andresen BS. Identification of SRSF10 as a regulator of SMN2 ISS-N1. Hum Mutat 2020; 42:246-260. [PMID: 33300159 DOI: 10.1002/humu.24149] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2020] [Revised: 10/22/2020] [Accepted: 12/06/2020] [Indexed: 01/02/2023]
Abstract
Understanding the splicing code can be challenging as several splicing factors bind to many splicing-regulatory elements. The SMN1 and SMN2 silencer element ISS-N1 is the target of the antisense oligonucleotide drug, Spinraza, which is the treatment against spinal muscular atrophy. However, limited knowledge about the nature of the splicing factors that bind to ISS-N1 and inhibit splicing exists. It is likely that the effect of Spinraza comes from blocking binding of these factors, but so far, an unbiased characterization has not been performed and only members of the hnRNP A1/A2 family have been identified by Western blot analysis and nuclear magnetic resonance to bind to this silencer. Employing an MS/MS-based approach and surface plasmon resonance imaging, we show for the first time that splicing factor SRSF10 binds to ISS-N1. Furthermore, using splice-switching oligonucleotides we modulated the splicing of the SRSF10 isoforms generating either the long or the short protein isoform of SRSF10 to regulate endogenous SMN2 exon 7 inclusion. We demonstrate that the isoforms of SRSF10 regulate SMN1 and SMN2 splicing with different strength correlating with the length of their RS domain. Our results suggest that the ratio between the SRSF10 isoforms is important for splicing regulation.
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Affiliation(s)
- Sabrina B Frederiksen
- Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark
| | - Lise L Holm
- Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark
| | - Martin R Larsen
- Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark
| | - Thomas K Doktor
- Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark
| | - Henriette S Andersen
- Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark
| | - Michelle L Hastings
- Department of Cell Biology and Anatomy, Center for Genetic Diseases, Chicago Medical School and School of Graduate and Postdoctoral Studies, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, USA
| | - Yimin Hua
- Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA
| | - Adrian R Krainer
- Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA
| | - Brage S Andresen
- Department of Biochemistry and Molecular Biology and the Villum Center for Bioanalytical Sciences, University of Southern Denmark, Odense M, Denmark
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16
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Kováčová T, Souček P, Hujová P, Freiberger T, Grodecká L. Splicing Enhancers at Intron-Exon Borders Participate in Acceptor Splice Sites Recognition. Int J Mol Sci 2020; 21:ijms21186553. [PMID: 32911621 PMCID: PMC7554774 DOI: 10.3390/ijms21186553] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2020] [Revised: 09/05/2020] [Accepted: 09/06/2020] [Indexed: 02/07/2023] Open
Abstract
Acceptor splice site recognition (3′ splice site: 3′ss) is a fundamental step in precursor messenger RNA (pre-mRNA) splicing. Generally, the U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor (U2AF) heterodimer recognizes the 3′ss, of which U2AF35 has a dual function: (i) It binds to the intron–exon border of some 3′ss and (ii) mediates enhancer-binding splicing activators’ interactions with the spliceosome. Alternative mechanisms for 3′ss recognition have been suggested, yet they are still not thoroughly understood. Here, we analyzed 3′ss recognition where the intron–exon border is bound by a ubiquitous splicing regulator SRSF1. Using the minigene analysis of two model exons and their mutants, BRCA2 exon 12 and VARS2 exon 17, we showed that the exon inclusion correlated much better with the predicted SRSF1 affinity than 3′ss quality, which were assessed using the Catalog of Inferred Sequence Binding Preferences of RNA binding proteins (CISBP-RNA) database and maximum entropy algorithm (MaxEnt) predictor and the U2AF35 consensus matrix, respectively. RNA affinity purification proved SRSF1 binding to the model 3′ss. On the other hand, knockdown experiments revealed that U2AF35 also plays a role in these exons’ inclusion. Most probably, both factors stochastically bind the 3′ss, supporting exon recognition, more apparently in VARS2 exon 17. Identifying splicing activators as 3′ss recognition factors is crucial for both a basic understanding of splicing regulation and human genetic diagnostics when assessing variants’ effects on splicing.
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Affiliation(s)
- Tatiana Kováčová
- Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation, 656 91 Brno, Czech Republic; (T.K.); (P.S.); (P.H.); (T.F.)
- Faculty of Medicine, Masaryk University, 625 00 Brno, Czech Republic
| | - Přemysl Souček
- Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation, 656 91 Brno, Czech Republic; (T.K.); (P.S.); (P.H.); (T.F.)
- Faculty of Medicine, Masaryk University, 625 00 Brno, Czech Republic
| | - Pavla Hujová
- Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation, 656 91 Brno, Czech Republic; (T.K.); (P.S.); (P.H.); (T.F.)
- Faculty of Medicine, Masaryk University, 625 00 Brno, Czech Republic
| | - Tomáš Freiberger
- Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation, 656 91 Brno, Czech Republic; (T.K.); (P.S.); (P.H.); (T.F.)
- Faculty of Medicine, Masaryk University, 625 00 Brno, Czech Republic
| | - Lucie Grodecká
- Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation, 656 91 Brno, Czech Republic; (T.K.); (P.S.); (P.H.); (T.F.)
- Correspondence:
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17
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Chen MX, Zhang KL, Gao B, Yang JF, Tian Y, Das D, Fan T, Dai L, Hao GF, Yang GF, Zhang J, Zhu FY, Fang YM. Phylogenetic comparison of 5' splice site determination in central spliceosomal proteins of the U1-70K gene family, in response to developmental cues and stress conditions. THE PLANT JOURNAL : FOR CELL AND MOLECULAR BIOLOGY 2020; 103:357-378. [PMID: 32133712 DOI: 10.1111/tpj.14735] [Citation(s) in RCA: 29] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/20/2018] [Revised: 01/22/2020] [Accepted: 02/04/2020] [Indexed: 05/07/2023]
Abstract
Intron-containing genes have the ability to generate multiple transcript isoforms by splicing, thereby greatly expanding the eukaryotic transcriptome and proteome. In eukaryotic cells, precursor mRNA (pre-mRNA) splicing is performed by a mega-macromolecular complex defined as a spliceosome. Among its splicing components, U1 small nuclear ribonucleoprotein (U1 snRNP) is the smallest subcomplex involved in early spliceosome assembly and 5'-splice site recognition. Its central component, named U1-70K, has been extensively characterized in animals and yeast. Very few investigations on U1-70K genes have been conducted in plants, however. To this end, we performed a comprehensive study to systematically identify 115 U1-70K genes from 67 plant species, ranging from algae to angiosperms. Phylogenetic analysis suggested that the expansion of the plant U1-70K gene family was likely to have been driven by whole-genome duplications. Subsequent comparisons of gene structures, protein domains, promoter regions and conserved splicing patterns indicated that plant U1-70Ks are likely to preserve their conserved molecular function across plant lineages and play an important functional role in response to environmental stresses. Furthermore, genetic analysis using T-DNA insertion mutants suggested that Arabidopsis U1-70K may be involved in response to osmotic stress. Our results provide a general overview of this gene family in Viridiplantae and will act as a reference source for future mechanistic studies on this U1 snRNP-specific splicing factor.
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Affiliation(s)
- Mo-Xian Chen
- Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Nanjing, 210037, China
- Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518063, China
- Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China
| | - Kai-Lu Zhang
- Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Nanjing, 210037, China
- Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China
| | - Bei Gao
- School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong
| | - Jing-Fang Yang
- Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, 430079, China
| | - Yuan Tian
- Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China
| | - Debatosh Das
- Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China
| | - Tao Fan
- Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China
| | - Lei Dai
- Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518063, China
| | - Ge-Fei Hao
- Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, 430079, China
| | - Guang-Fu Yang
- Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, 430079, China
| | - Jianhua Zhang
- Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China
- Department of Biology, Hong Kong Baptist University, Shatin, Hong Kong
- State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, Hong Kong
| | - Fu-Yuan Zhu
- Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Nanjing, 210037, China
| | - Yan-Ming Fang
- Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Nanjing, 210037, China
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18
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Mineo M, Lyons SM, Zdioruk M, von Spreckelsen N, Ferrer-Luna R, Ito H, Alayo QA, Kharel P, Giantini Larsen A, Fan WY, Auduong S, Grauwet K, Passaro C, Khalsa JK, Shah K, Reardon DA, Ligon KL, Beroukhim R, Nakashima H, Ivanov P, Anderson PJ, Lawler SE, Chiocca EA. Tumor Interferon Signaling Is Regulated by a lncRNA INCR1 Transcribed from the PD-L1 Locus. Mol Cell 2020; 78:1207-1223.e8. [PMID: 32504554 DOI: 10.1016/j.molcel.2020.05.015] [Citation(s) in RCA: 47] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2019] [Revised: 03/03/2020] [Accepted: 05/11/2020] [Indexed: 01/22/2023]
Abstract
Tumor interferon (IFN) signaling promotes PD-L1 expression to suppress T cell-mediated immunosurveillance. We identify the IFN-stimulated non-coding RNA 1 (INCR1) as a long noncoding RNA (lncRNA) transcribed from the PD-L1 locus and show that INCR1 controls IFNγ signaling in multiple tumor types. Silencing INCR1 decreases the expression of PD-L1, JAK2, and several other IFNγ-stimulated genes. INCR1 knockdown sensitizes tumor cells to cytotoxic T cell-mediated killing, improving CAR T cell therapy. We discover that PD-L1 and JAK2 transcripts are negatively regulated by binding to HNRNPH1, a nuclear ribonucleoprotein. The primary transcript of INCR1 binds HNRNPH1 to block its inhibitory effects on the neighboring genes PD-L1 and JAK2, enabling their expression. These findings introduce a mechanism of tumor IFNγ signaling regulation mediated by the lncRNA INCR1 and suggest a therapeutic target for cancer immunotherapy.
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Affiliation(s)
- Marco Mineo
- Harvey W. Cushing Neuro-oncology Laboratories (HCNL), Department of Neurosurgery, Harvard Medical School and Brigham and Women's Hospital, Boston, MA 02115, USA.
| | - Shawn M Lyons
- Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA
| | - Mykola Zdioruk
- Harvey W. Cushing Neuro-oncology Laboratories (HCNL), Department of Neurosurgery, Harvard Medical School and Brigham and Women's Hospital, Boston, MA 02115, USA
| | - Niklas von Spreckelsen
- Harvey W. Cushing Neuro-oncology Laboratories (HCNL), Department of Neurosurgery, Harvard Medical School and Brigham and Women's Hospital, Boston, MA 02115, USA; Department of Neurosurgery, Center for Neurosurgery, Faculty of Medicine, and University Hospital, University of Cologne, 50937 Cologne, Germany
| | - Ruben Ferrer-Luna
- Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Cancer Program, The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA
| | - Hirotaka Ito
- Harvey W. Cushing Neuro-oncology Laboratories (HCNL), Department of Neurosurgery, Harvard Medical School and Brigham and Women's Hospital, Boston, MA 02115, USA
| | - Quazim A Alayo
- Harvey W. Cushing Neuro-oncology Laboratories (HCNL), Department of Neurosurgery, Harvard Medical School and Brigham and Women's Hospital, Boston, MA 02115, USA
| | - Prakash Kharel
- Division of Rheumatology, Inflammation, and Immunity, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
| | - Alexandra Giantini Larsen
- Harvey W. Cushing Neuro-oncology Laboratories (HCNL), Department of Neurosurgery, Harvard Medical School and Brigham and Women's Hospital, Boston, MA 02115, USA
| | - William Y Fan
- Harvey W. Cushing Neuro-oncology Laboratories (HCNL), Department of Neurosurgery, Harvard Medical School and Brigham and Women's Hospital, Boston, MA 02115, USA
| | - Sophia Auduong
- Harvey W. Cushing Neuro-oncology Laboratories (HCNL), Department of Neurosurgery, Harvard Medical School and Brigham and Women's Hospital, Boston, MA 02115, USA
| | - Korneel Grauwet
- Harvey W. Cushing Neuro-oncology Laboratories (HCNL), Department of Neurosurgery, Harvard Medical School and Brigham and Women's Hospital, Boston, MA 02115, USA
| | - Carmela Passaro
- Harvey W. Cushing Neuro-oncology Laboratories (HCNL), Department of Neurosurgery, Harvard Medical School and Brigham and Women's Hospital, Boston, MA 02115, USA
| | - Jasneet K Khalsa
- Center for Stem Cell Therapeutics and Imaging, Department of Neurosurgery, Brigham and Women's Hospital, Boston, MA 02115, USA
| | - Khalid Shah
- Center for Stem Cell Therapeutics and Imaging, Department of Neurosurgery, Brigham and Women's Hospital, Boston, MA 02115, USA
| | - David A Reardon
- Center for Neuro-Oncology, Dana-Farber Cancer Institute, and Brigham and Women's Hospital, Boston, MA 02115, USA
| | - Keith L Ligon
- Cancer Program, The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Oncologic Pathology, Dana-Farber Cancer Institute, Harvard Medical School, Boston Children's Hospital, and Brigham and Women's Hospital, Boston, MA 02115, USA
| | - Rameen Beroukhim
- Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA; Cancer Program, The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Center for Neuro-Oncology, Dana-Farber Cancer Institute, and Brigham and Women's Hospital, Boston, MA 02115, USA
| | - Hiroshi Nakashima
- Harvey W. Cushing Neuro-oncology Laboratories (HCNL), Department of Neurosurgery, Harvard Medical School and Brigham and Women's Hospital, Boston, MA 02115, USA
| | - Pavel Ivanov
- Division of Rheumatology, Inflammation, and Immunity, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Harvard Medical School Initiative for RNA Medicine, Boston, MA 02115, USA
| | - Paul J Anderson
- Division of Rheumatology, Inflammation, and Immunity, Brigham and Women's Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115, USA; Harvard Medical School Initiative for RNA Medicine, Boston, MA 02115, USA
| | - Sean E Lawler
- Harvey W. Cushing Neuro-oncology Laboratories (HCNL), Department of Neurosurgery, Harvard Medical School and Brigham and Women's Hospital, Boston, MA 02115, USA
| | - E Antonio Chiocca
- Harvey W. Cushing Neuro-oncology Laboratories (HCNL), Department of Neurosurgery, Harvard Medical School and Brigham and Women's Hospital, Boston, MA 02115, USA.
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19
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Tiek DM, Khatib SA, Trepicchio CJ, Heckler MM, Divekar SD, Sarkaria JN, Glasgow E, Riggins RB. Estrogen-related receptor β activation and isoform shifting by cdc2-like kinase inhibition restricts migration and intracranial tumor growth in glioblastoma. FASEB J 2019; 33:13476-13491. [PMID: 31570001 PMCID: PMC6894094 DOI: 10.1096/fj.201901075r] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2019] [Accepted: 08/26/2019] [Indexed: 11/11/2022]
Abstract
Glioblastoma (GBM; grade 4 glioma) is a highly aggressive and incurable tumor. GBM has recently been characterized as highly dependent on alternative splicing, a critical driver of tumor heterogeneity and plasticity. Estrogen-related receptor β (ERR-β) is an orphan nuclear receptor expressed in the brain, where alternative splicing of the 3' end of the pre-mRNA leads to the production of 3 validated ERR-β protein products: ERR-β short form (ERR-βsf), ERR-β2, and ERR-β exon 10 deleted. Our prior studies have shown the ERR-β2 isoform to play a role in G2/M cell cycle arrest and induction of apoptosis, in contrast to the function of the shorter ERR-βsf isoform in senescence and G1 cell cycle arrest. In this study, we sought to better define the role of the proapoptotic ERR-β2 isoform in GBM. We show that the ERR-β2 isoform is located not only in the nucleus but also in the cytoplasm. ERR-β2 suppresses GBM cell migration and interacts with the actin nucleation-promoting factor cortactin, and an ERR-β agonist is able to remodel the actin cytoskeleton and similarly suppress GBM cell migration. We further show that inhibition of the splicing regulatory cdc2-like kinases in combination with an ERR-β agonist shifts isoform expression in favor of ERR-β2 and potentiates inhibition of growth and migration in GBM cells and intracranial tumors.-Tiek, D. M., Khatib, S. A., Trepicchio, C. J., Heckler, M. M., Divekar, S. D., Sarkaria, J. N., Glasgow, E., Riggins, R. B. Estrogen-related receptor β activation and isoform shifting by cdc2-like kinase inhibition restricts migration and intracranial tumor growth in glioblastoma.
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Affiliation(s)
- Deanna M. Tiek
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA
| | - Subreen A. Khatib
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA
- Laboratory of Human Carcinogenesis, National Cancer Institute, Bethesda, Maryland, USA; and
| | - Colin J. Trepicchio
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA
| | - Mary M. Heckler
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA
| | - Shailaja D. Divekar
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA
| | - Jann N. Sarkaria
- Department of Radiation Oncology, Mayo Clinic, Rochester, Minnesota, USA
| | - Eric Glasgow
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA
| | - Rebecca B. Riggins
- Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA
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20
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Zhu Y, Deng H, Chen X, Li H, Yang C, Li S, Pan X, Tian S, Feng S, Tan X, Matsuo M, Zhang Z. Skipping of an exon with a nonsense mutation in the DMD gene is induced by the conversion of a splicing enhancer to a splicing silencer. Hum Genet 2019; 138:771-785. [DOI: 10.1007/s00439-019-02036-2] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2019] [Accepted: 05/29/2019] [Indexed: 01/23/2023]
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21
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De Franco S, Vandenameele J, Brans A, Verlaine O, Bendak K, Damblon C, Matagne A, Segal DJ, Galleni M, Mackay JP, Vandevenne M. Exploring the suitability of RanBP2-type Zinc Fingers for RNA-binding protein design. Sci Rep 2019; 9:2484. [PMID: 30792407 PMCID: PMC6384913 DOI: 10.1038/s41598-019-38655-y] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2018] [Accepted: 11/30/2018] [Indexed: 12/16/2022] Open
Abstract
Transcriptomes consist of several classes of RNA that have wide-ranging but often poorly described functions and the deregulation of which leads to numerous diseases. Engineering of functionalized RNA-binding proteins (RBPs) could therefore have many applications. Our previous studies suggested that the RanBP2-type Zinc Finger (ZF) domain is a suitable scaffold to investigate the design of single-stranded RBPs. In the present work, we have analyzed the natural sequence specificity of various members of the RanBP2-type ZF family and characterized the interaction with their target RNA. Surprisingly, our data showed that natural RanBP2-type ZFs with different RNA-binding residues exhibit a similar sequence specificity and therefore no simple recognition code can be established. Despite this finding, different discriminative abilities were observed within the family. In addition, in order to target a long RNA sequence and therefore gain in specificity, we generated a 6-ZF array by combining ZFs from the RanBP2-type family but also from different families, in an effort to achieve a wider target sequence repertoire. We showed that this chimeric protein recognizes its target sequence (20 nucleotides), both in vitro and in living cells. Altogether, our results indicate that the use of ZFs in RBP design remains attractive even though engineering of specificity changes is challenging.
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Affiliation(s)
- Simona De Franco
- InBioS-Centre d'Ingénierie des Protéines (CIP), Université de Liège, Liège, 4000, Belgium
| | - Julie Vandenameele
- InBioS-Centre d'Ingénierie des Protéines (CIP), Université de Liège, Liège, 4000, Belgium
| | - Alain Brans
- InBioS-Centre d'Ingénierie des Protéines (CIP), Université de Liège, Liège, 4000, Belgium
| | - Olivier Verlaine
- InBioS-Centre d'Ingénierie des Protéines (CIP), Université de Liège, Liège, 4000, Belgium
| | - Katerina Bendak
- Children's Cancer Institute Lowy Cancer Research, Kensington, 2033, Australia
| | - Christian Damblon
- Laboratoire de Chimie Biologique Structurale (CBS), Département de Chimie, Université de Liège, Liège, 4000, Belgium
| | - André Matagne
- InBioS-Centre d'Ingénierie des Protéines (CIP), Université de Liège, Liège, 4000, Belgium
| | - David J Segal
- Genome Center and Department of Biochemistry and Molecular Medicine, University of California, Davis, CA, 95616, USA
| | - Moreno Galleni
- InBioS-Centre d'Ingénierie des Protéines (CIP), Université de Liège, Liège, 4000, Belgium.
| | - Joel P Mackay
- School of Life and Environmental Sciences, University of Sydney, Sydney, N.S.W, 2006, Australia
| | - Marylène Vandevenne
- InBioS-Centre d'Ingénierie des Protéines (CIP), Université de Liège, Liège, 4000, Belgium.
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22
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Majoros WH, Holt C, Campbell MS, Ware D, Yandell M, Reddy TE. Predicting gene structure changes resulting from genetic variants via exon definition features. Bioinformatics 2018; 34:3616-3623. [PMID: 29701825 PMCID: PMC6198862 DOI: 10.1093/bioinformatics/bty324] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2018] [Revised: 03/28/2018] [Accepted: 04/24/2018] [Indexed: 01/24/2023] Open
Abstract
Motivation Genetic variation that disrupts gene function by altering gene splicing between individuals can substantially influence traits and disease. In those cases, accurately predicting the effects of genetic variation on splicing can be highly valuable for investigating the mechanisms underlying those traits and diseases. While methods have been developed to generate high quality computational predictions of gene structures in reference genomes, the same methods perform poorly when used to predict the potentially deleterious effects of genetic changes that alter gene splicing between individuals. Underlying that discrepancy in predictive ability are the common assumptions by reference gene finding algorithms that genes are conserved, well-formed and produce functional proteins. Results We describe a probabilistic approach for predicting recent changes to gene structure that may or may not conserve function. The model is applicable to both coding and non-coding genes, and can be trained on existing gene annotations without requiring curated examples of aberrant splicing. We apply this model to the problem of predicting altered splicing patterns in the genomes of individual humans, and we demonstrate that performing gene-structure prediction without relying on conserved coding features is feasible. The model predicts an unexpected abundance of variants that create de novo splice sites, an observation supported by both simulations and empirical data from RNA-seq experiments. While these de novo splice variants are commonly misinterpreted by other tools as coding or non-coding variants of little or no effect, we find that in some cases they can have large effects on splicing activity and protein products and we propose that they may commonly act as cryptic factors in disease. Availability and implementation The software is available from geneprediction.org/SGRF. Supplementary information Supplementary information is available at Bioinformatics online.
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Affiliation(s)
- William H Majoros
- Program in Computational Biology and Bioinformatics, Duke University, Durham, NC, USA
- Center for Genomic and Computational Biology, Duke University Medical School, Durham, NC, USA
| | - Carson Holt
- Department of Human Genetics, Eccles Institute of Human Genetics, University of Utah and School of Medicine, Salt Lake City, UT, USA
- USTAR Center for Genetic Discovery, University of Utah, Salt Lake City, UT, USA
| | | | - Doreen Ware
- Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
- USDA ARS NEA Robert W. Holley Center for Agriculture and Health, Cornell University, Ithaca, NY, USA
| | - Mark Yandell
- Department of Human Genetics, Eccles Institute of Human Genetics, University of Utah and School of Medicine, Salt Lake City, UT, USA
- USTAR Center for Genetic Discovery, University of Utah, Salt Lake City, UT, USA
| | - Timothy E Reddy
- Program in Computational Biology and Bioinformatics, Duke University, Durham, NC, USA
- Center for Genomic and Computational Biology, Duke University Medical School, Durham, NC, USA
- Department of Biostatistics and Bioinformatics, Duke University Medical School, Durham, NC, USA
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23
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QKI5-mediated alternative splicing of the histone variant macroH2A1 regulates gastric carcinogenesis. Oncotarget 2017; 7:32821-34. [PMID: 27092877 PMCID: PMC5078054 DOI: 10.18632/oncotarget.8739] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2015] [Accepted: 03/28/2016] [Indexed: 12/18/2022] Open
Abstract
Alternative pre-mRNA splicing is a key mechanism for increasing proteomic diversity and modulating gene expression. Emerging evidence indicated that the splicing program is frequently dysregulated during tumorigenesis. Cancer cells produce protein isoforms that can promote growth and survival. The RNA-binding protein QKI5 is a critical regulator of alternative splicing in expanding lists of primary human tumors and tumor cell lines. However, its biological role and regulatory mechanism are poorly defined in gastric cancer (GC) development and progression. In this study, we demonstrated that the downregulation of QKI5 was associated with pTNM stage and pM state of GC patients. Re-introduction of QKI5 could inhibit GC cell proliferation, migration, and invasion in vitro and in vivo, which might be due to the altered splicing pattern of macroH2A1 pre-mRNA, leading to the accumulation of macroH2A1.1 isoform. Furthermore, QKI5 could inhibit cyclin L1 expression via promoting macroH2A1.1 production. Thus, this study identified a novel regulatory axis involved in gastric tumorigenesis and provided a new strategy for GC therapy.
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24
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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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25
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Ohno K, Takeda JI, Masuda A. Rules and tools to predict the splicing effects of exonic and intronic mutations. WILEY INTERDISCIPLINARY REVIEWS-RNA 2017; 9. [DOI: 10.1002/wrna.1451] [Citation(s) in RCA: 71] [Impact Index Per Article: 8.9] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/26/2017] [Revised: 08/23/2017] [Accepted: 08/24/2017] [Indexed: 12/14/2022]
Affiliation(s)
- Kinji Ohno
- Division of Neurogenetics, Center for Neurological Diseases and Cancer; Nagoya University Graduate School of Medicine; Nagoya Japan
| | - Jun-ichi Takeda
- Division of Neurogenetics, Center for Neurological Diseases and Cancer; Nagoya University Graduate School of Medicine; Nagoya Japan
| | - Akio Masuda
- Division of Neurogenetics, Center for Neurological Diseases and Cancer; Nagoya University Graduate School of Medicine; Nagoya Japan
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26
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SRSF1 suppresses selection of intron-distal 5' splice site of DOK7 intron 4 to generate functional full-length Dok-7 protein. Sci Rep 2017; 7:10446. [PMID: 28874828 PMCID: PMC5585400 DOI: 10.1038/s41598-017-11036-z] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2017] [Accepted: 08/18/2017] [Indexed: 01/23/2023] Open
Abstract
Dok-7 is a non-catalytic adaptor protein that facilitates agrin-induced clustering of acetylcholine receptors (AChR) at the neuromuscular junction. Alternative selection of 5′ splice sites (SSs) of DOK7 intron 4 generates canonical and frame-shifted transcripts. We found that the canonical full-length Dok-7 enhanced AChR clustering, whereas the truncated Dok-7 did not. We identified a splicing cis-element close to the 3′ end of exon 4 by block-scanning mutagenesis. RNA affinity purification and mass spectrometry revealed that SRSF1 binds to the cis-element. Knocking down of SRSF1 enhanced selection of the intron-distal 5′ SS of DOK7 intron 4, whereas MS2-mediated artificial tethering of SRSF1 to the identified cis-element suppressed it. Isolation of an early spliceosomal complex revealed that SRSF1 inhibited association of U1 snRNP to the intron-distal 5′ SS, and rather enhanced association of U1 snRNP to the intron-proximal 5′ SS, which led to upregulation of the canonical DOK7 transcript. Integrated global analysis of CLIP-seq and RNA-seq also indicated that binding of SRSF1 immediately upstream to two competing 5′ SSs suppresses selection of the intron-distal 5′ SS in hundreds of human genes. We demonstrate that SRSF1 critically regulates alternative selection of adjacently placed 5′ SSs by modulating binding of U1 snRNP.
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27
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Pathogenic variants that alter protein code often disrupt splicing. Nat Genet 2017; 49:848-855. [PMID: 28416821 PMCID: PMC6679692 DOI: 10.1038/ng.3837] [Citation(s) in RCA: 168] [Impact Index Per Article: 21.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2016] [Accepted: 03/16/2017] [Indexed: 12/18/2022]
Abstract
The lack of tools to identify causative variants from sequencing data greatly limits the promise of Precision Medicine. Previous studies suggest one-third of disease alleles alter splicing. We discovered that splicing defects cluster in diseases (e.g. haploinsufficient genes). We analyzed 4,964 published disease-causing exonic mutations using a Massively Parallel Splicing Assay (MaPSy) that showed 81% concordance rate with patient tissue splicing. ~10% of exonic mutations altered splicing, mostly by disrupting multiple stages of the spliceosome assembly. We present the first large-scale characterization of exonic splicing mutations using a novel technology that facilitates variant classification that keeps pace with variant discovery.
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28
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Ohno K, Rahman MA, Nazim M, Nasrin F, Lin Y, Takeda JI, Masuda A. Splicing regulation and dysregulation of cholinergic genes expressed at the neuromuscular junction. J Neurochem 2017; 142 Suppl 2:64-72. [PMID: 28072465 DOI: 10.1111/jnc.13954] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2016] [Revised: 12/23/2016] [Accepted: 01/02/2017] [Indexed: 02/05/2023]
Abstract
We humans have evolved by acquiring diversity of alternative RNA metabolisms including alternative means of splicing and transcribing non-coding genes, and not by acquiring new coding genes. Tissue-specific and developmental stage-specific alternative RNA splicing is achieved by tightly regulated spatiotemporal regulation of expressions and activations of RNA-binding proteins that recognize their cognate splicing cis-elements on nascent RNA transcripts. Genes expressed at the neuromuscular junction are also alternatively spliced. In addition, germline mutations provoke aberrant splicing by compromising binding of RNA-binding proteins, and cause congenital myasthenic syndromes (CMS). We present physiological splicing mechanisms of genes for agrin (AGRN), acetylcholinesterase (ACHE), MuSK (MUSK), acetylcholine receptor (AChR) α1 subunit (CHRNA1), and collagen Q (COLQ) in human, and their aberration in diseases. Splicing isoforms of AChET , AChEH , and AChER are generated by hnRNP H/F. Skipping of MUSK exon 10 makes a Wnt-insensitive MuSK isoform, which is unique to human. Skipping of exon 10 is achieved by coordinated binding of hnRNP C, YB-1, and hnRNP L to exon 10. Exon P3A of CHRNA1 is alternatively included to generate a non-functional AChR α1 subunit in human. Molecular dissection of splicing mutations in patients with CMS reveals that exon P3A is alternatively skipped by hnRNP H, polypyrimidine tract-binding protein 1, and hnRNP L. Similarly, analysis of an exonic mutation in COLQ exon 16 in a CMS patient discloses that constitutive splicing of exon 16 requires binding of serine arginine-rich splicing factor 1. Intronic and exonic splicing mutations in CMS enable us to dissect molecular mechanisms underlying alternative and constitutive splicing of genes expressed at the neuromuscular junction. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.
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Affiliation(s)
- Kinji Ohno
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Mohammad Alinoor Rahman
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Mohammad Nazim
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Farhana Nasrin
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Yingni Lin
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Jun-Ichi Takeda
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan
| | - Akio Masuda
- Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan
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Silipo M, Gautrey H, Satam S, Lennard T, Tyson-Capper A. How is Herstatin, a tumor suppressor splice variant of the oncogene HER2, regulated? RNA Biol 2016; 14:536-543. [PMID: 27935425 DOI: 10.1080/15476286.2016.1267074] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The human epidermal growth factor receptor 2 (HER2)/receptor tyrosine-protein kinasebB-2 (ERBB2) is overexpressed in 20-30% of breast tumors leading to faster growing and more aggressive tumors. Alternative splicing generates a functionally distinct HER2 variant called Herstatin, which is produced by the inclusion of intron 8. Herstatin acts as a tumor suppressor by effectively blocking HER2 activity and cell proliferation, while promoting apoptosis. In the present study we investigated HER2 pre-mRNA regulatory sequences and splicing factors which regulate the alternative splicing of Herstatin. A Herstatin minigene, comprising exon 8/intron 8/exon 9 of HER2 was generated and subsequent in vitro splicing assays revealed that RNA secondary structure and somatic mutations did not impact on inclusion of intron 8. However, using RNase-assisted RNA chromatography, followed by mass spectrometry, we identified six RNA-binding proteins (splicing factors) that bind to RNA sequences surrounding exon 8/intron 8 and intron 8/exon 9 boundaries; these included hnRNP I, H1, D, A2/B1 and hnRNPA1 plus the SR protein SRSF1. Specifically, overexpression of hnRNP A1 significantly increased retention of intron 8 resulting in higher levels of Herstatin in SKBR3 breast cancer cells whereas SRSF1 only had a marginal effect in decreasing Herstatin but increased exogenous HER2 levels under these experimental conditions. In conclusion, we have identified the first splicing factors and regulatory sequences that are involved in the production of Herstatin.
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Affiliation(s)
- Marco Silipo
- a Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University , Newcastle , UK
| | - Hannah Gautrey
- a Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University , Newcastle , UK
| | - Swapna Satam
- a Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University , Newcastle , UK
| | - Thomas Lennard
- b Northern Institute for Cancer Research, Faculty of Medical Sciences, Newcastle University , Newcastle , UK
| | - Alison Tyson-Capper
- a Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University , Newcastle , UK
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30
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Sun YL, Liu F, Liu F, Zhao XH. Protein and gene expression characteristics of heterogeneous nuclear ribonucleoprotein H1 in esophageal squamous cell carcinoma. World J Gastroenterol 2016; 22:7322-7331. [PMID: 27621578 PMCID: PMC4997634 DOI: 10.3748/wjg.v22.i32.7322] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/26/2016] [Revised: 05/16/2016] [Accepted: 06/02/2016] [Indexed: 02/06/2023] Open
Abstract
AIM To investigate the expression characteristics of heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) mRNA and protein in cell lines and tissues of esophageal squamous cell carcinoma (ESCC).
METHODS Western blotting was used to assess the expression of HNRNPH1 protein in seven ESCC cell lines and 30 paired fresh tissue specimens. The subcellular localization of HNRNPH1 was determined by immunofluorescence in ESCC cells. The RNA sequencing data from 87 patients with ESCC were obtained from the cancer genome atlas (TCGA), and the expression and clinical characteristics analysis of different transcript variants of HNRNPH1 were evaluated in this dataset. In addition, immunohistochemistry was carried out to detect the expression of HNRNPH1 protein in 125 patients.
RESULTS The expression of HNRNPH1 protein varied across different ESCC cell lines. It was exclusively restricted to the nucleus of the ESCC cells. There are two transcript variants of the HNRNPH1 gene. Variant 1 was constitutively expressed, and its expression did not change during tumorigenesis. In contrast, levels of variant 2 were low in non-tumorous tissues and were dramatically increased in ESCC (P = 0.0026). The high levels of variant 2 were associated with poorer differentiated tumors (P = 0.0287). Furthermore, in paired fresh tissue specimens, HNRNPH1 protein was overexpressed in 73.3% (22/30) of neoplastic tissues. HNRNPH1 was significantly upregulated in ESCC, with strong staining in 43.2% (54/125) of tumor tissues and 22.4% (28/125) of matched non-cancerous tissues (P = 0.0005). Positive HNRNPH1 expression was significantly associated with poor tumor differentiation degree (P = 0.0337).
CONCLUSION The different alternative transcript variants of HNRNPH1 exhibited different expression changes during tumorigenesis. Its mRNA and protein were overexpressed in ESCC and associated with poorer differentiation of tumor cells. These findings highlight the potential of HNRNPH1 in the therapy and diagnosis of ESCC.
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MESH Headings
- Adult
- Aged
- Alternative Splicing
- Apoptosis
- Biomarkers, Tumor/genetics
- Biomarkers, Tumor/metabolism
- Carcinoma, Squamous Cell/genetics
- Carcinoma, Squamous Cell/metabolism
- Carcinoma, Squamous Cell/pathology
- Cell Line, Tumor
- Cell Proliferation
- Esophageal Neoplasms/genetics
- Esophageal Neoplasms/metabolism
- Esophageal Neoplasms/pathology
- Esophageal Squamous Cell Carcinoma
- Female
- Gene Expression Regulation, Neoplastic
- Heterogeneous-Nuclear Ribonucleoprotein Group F-H/genetics
- Heterogeneous-Nuclear Ribonucleoprotein Group F-H/metabolism
- Humans
- Male
- Middle Aged
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- RNA, Neoplasm/genetics
- RNA, Neoplasm/metabolism
- Up-Regulation
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