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Hou A, Xu X, Zhang Y, He H, Feng Y, Fan W, Tan R, Gong L, Chen J. Excessive fatty acids activate PRMT5/MDM2/Drosha pathway to regulate miRNA biogenesis and lipid metabolism. Liver Int 2024; 44:1634-1650. [PMID: 38517158 DOI: 10.1111/liv.15906] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/05/2023] [Revised: 02/26/2024] [Accepted: 03/07/2024] [Indexed: 03/23/2024]
Abstract
BACKGROUND Excessive fatty acids in the liver lead to the accumulation of lipotoxic lipids and then cellular stress to further evoke the related disease, like non-alcoholic fatty liver disease (NAFLD). As reported, fatty acid stimulation can cause some specific miRNA dysregulation, which caused us to investigate the relationship between miRNA biogenesis and fatty acid overload. METHODS Gene expression omnibus (GEO) dataset analysis, miRNA-seq, miRNA cleavage assay, RT-qPCR, western blotting, immunofluorescence and co-immunoprecipitation (co-IP) were used to reveal the change of miRNAs under pathological status and explore the relevant mechanism. High fat, high fructose, high cholesterol (HFHFrHC) diet-fed mice transfected with AAV2/8-shDrosha or AAV2/8-shPRMT5 were established to investigate the in vivo effects of Drosha or PRMT5 on NAFLD phenotype. RESULTS We discovered that the cleavage of miRNAs was inhibited by analysing miRNA contents and detecting some representative pri-miRNAs in multiple mouse and cell models, which was further verified by the reduction of the Microprocessor activity in the presence of palmitic acid (PA). In vitro, PA could induce Drosha, the core RNase III in the Microprocessor complex, degrading through the proteasome-mediated pathway, while in vivo, knockdown of Drosha significantly promoted NAFLD to develop to a more serious stage. Mechanistically, our results demonstrated that PA can increase the methyltransferase activity of PRMT5 to degrade Drosha through MDM2, a ubiquitin E3 ligase for Drosha. The above results indicated that PRMT5 may be a critical regulator in lipid metabolism during NAFLD, which was confirmed by the knocking down of PRMT5 improved aberrant lipid metabolism in vitro and in vivo. CONCLUSIONS We first demonstrated the relationship between miRNA dosage and NAFLD and proved that PA can activate the PRMT5-MDM2-Drosha signalling pathway to regulate miRNA biogenesis.
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Affiliation(s)
- Aijun Hou
- Center for Drug Safety Evaluation and Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
- School of Pharmacy, University of Chinese Academy of Sciences, Beijing, China
| | - Xiaoding Xu
- Center for Drug Safety Evaluation and Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
- School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China
| | - Yu Zhang
- Center for Drug Safety Evaluation and Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
- School of Pharmacy, University of Chinese Academy of Sciences, Beijing, China
| | - Hongxiu He
- Center for Drug Safety Evaluation and Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
- School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China
| | - Yihan Feng
- Department of Pharmaceutics, School of Pharmacy, Shenyang Pharmaceutical University, Shenyang, China
| | - Wenhui Fan
- Center for Drug Safety Evaluation and Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
- School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, China
| | - Rongrong Tan
- Center for Drug Safety Evaluation and Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
| | - Likun Gong
- Center for Drug Safety Evaluation and Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
- School of Pharmacy, University of Chinese Academy of Sciences, Beijing, China
| | - Jing Chen
- Center for Drug Safety Evaluation and Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
- School of Pharmacy, University of Chinese Academy of Sciences, Beijing, China
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2
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Gu K, Mok L, Wakefield MJ, Chong MMW. Non-canonical RNA substrates of Drosha lack many of the conserved features found in primary microRNA stem-loops. Sci Rep 2024; 14:6713. [PMID: 38509178 PMCID: PMC10954719 DOI: 10.1038/s41598-024-57330-5] [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: 11/12/2023] [Accepted: 03/18/2024] [Indexed: 03/22/2024] Open
Abstract
The RNase III enzyme Drosha has a central role in microRNA (miRNA) biogenesis, where it is required to release the stem-loop intermediate from primary (pri)-miRNA transcripts. However, it can also cleave stem-loops embedded within messenger (m)RNAs. This destabilizes the mRNA causing target gene repression and appears to occur primarily in stem cells. While pri-miRNA stem-loops have been extensively studied, such non-canonical substrates of Drosha have yet to be characterized in detail. In this study, we employed high-throughput sequencing to capture all polyA-tailed RNAs that are cleaved by Drosha in mouse embryonic stem cells (ESCs) and compared the features of non-canonical versus miRNA stem-loop substrates. mRNA substrates are less efficiently processed than miRNA stem-loops. Sequence and structural analyses revealed that these mRNA substrates are also less stable and more likely to fold into alternative structures than miRNA stem-loops. Moreover, they lack the sequence and structural motifs found in miRNA stem-loops that are required for precise cleavage. Notably, we discovered a non-canonical Drosha substrate that is cleaved in an inverse manner, which is a process that is normally inhibited by features in miRNA stem-loops. Our study thus provides valuable insights into the recognition of non-canonical targets by Drosha.
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Affiliation(s)
- Karen Gu
- St Vincent's Institute of Medical Research, Fitzroy, VIC, 3065, Australia
- Department of Medicine (St Vincent's), University of Melbourne, Fitzroy, VIC, 3065, Australia
| | - Lawrence Mok
- St Vincent's Institute of Medical Research, Fitzroy, VIC, 3065, Australia
| | - Matthew J Wakefield
- Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, 3052, Australia
- Department of Obstetrics and Gynaecology, University of Melbourne, Parkville, VIC, 3010, Australia
| | - Mark M W Chong
- St Vincent's Institute of Medical Research, Fitzroy, VIC, 3065, Australia.
- Department of Medicine (St Vincent's), University of Melbourne, Fitzroy, VIC, 3065, Australia.
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Prabhakar A, Hu S, Tang J, Ghatpande P, Lagna G, Jiang X, Hata A. Essential role of the amino-terminal region of Drosha for the Microprocessor function. iScience 2023; 26:107971. [PMID: 37810246 PMCID: PMC10558778 DOI: 10.1016/j.isci.2023.107971] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2022] [Revised: 03/06/2023] [Accepted: 09/15/2023] [Indexed: 10/10/2023] Open
Abstract
Drosha is a core component of the Microprocessor complex that cleaves primary-microRNAs (pri-miRNAs) to generate precursor-miRNA and regulates the expression of ∼80 ribosomal protein (RP) genes. Despite the fact that mutations in the amino-terminal region of Drosha (Drosha-NTR) are associated with a vascular disorder, hereditary hemorrhagic telangiectasia, the precise function of Drosha-NTR remains unclear. By deleting exon 5 from the Drosha gene and generating a Drosha mutant lacking the NTR (ΔN), we demonstrate that ΔN is unable to process pri-miRNAs, which leads to a global miRNA depletion, except for the miR-183/96/182 cluster. We find that Argonaute 2 facilitates the processing of the pri-miR-183/96/182 in ΔN cells. Unlike full-length Drosha, ΔN is not degraded under serum starvation, resulting in unregulated RP biogenesis and protein synthesis in ΔN cells, allowing them to evade growth arrest. This study reveals the essential role of Drosha-NTR in miRNA production and nutrient-dependent translational control.
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Affiliation(s)
- Amit Prabhakar
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Song Hu
- Molecular Cancer Research Center, Sun Yat-Sen University School of Medicine, Guangzhou 511400, P.R.China
| | - Jin Tang
- Molecular Cancer Research Center, Sun Yat-Sen University School of Medicine, Guangzhou 511400, P.R.China
| | - Prajakta Ghatpande
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Giorgio Lagna
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA
- Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94143, USA
| | - Xuan Jiang
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA
- Molecular Cancer Research Center, Sun Yat-Sen University School of Medicine, Guangzhou 511400, P.R.China
| | - Akiko Hata
- Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94143, USA
- Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143, USA
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4
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Son S, Kim B, Yang J, Kim VN. Role of the proline-rich disordered domain of DROSHA in intronic microRNA processing. Genes Dev 2023; 37:383-397. [PMID: 37236670 PMCID: PMC10270192 DOI: 10.1101/gad.350275.122] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2022] [Accepted: 04/24/2023] [Indexed: 05/28/2023]
Abstract
DROSHA serves as a gatekeeper of the microRNA (miRNA) pathway by processing primary transcripts (pri-miRNAs). While the functions of structured domains of DROSHA have been well documented, the contribution of N-terminal proline-rich disordered domain (PRD) remains elusive. Here we show that the PRD promotes the processing of miRNA hairpins located within introns. We identified a DROSHA isoform (p140) lacking the PRD, which is produced by proteolytic cleavage. Small RNA sequencing revealed that p140 is significantly impaired in the maturation of intronic miRNAs. Consistently, our minigene constructs demonstrated that PRD enhances the processing of intronic hairpins, but not those in exons. Splice site mutations did not affect the PRD's enhancing effect on intronic constructs, suggesting that the PRD acts independently of splicing reaction by interacting with sequences residing within introns. The N-terminal regions from zebrafish and Xenopus DROSHA can replace the human counterpart, indicating functional conservation despite poor sequence alignment. Moreover, we found that rapidly evolving intronic miRNAs are generally more dependent on PRD than conserved ones, suggesting a role of PRD in miRNA evolution. Our study reveals a new layer of miRNA regulation mediated by a low-complexity disordered domain that senses the genomic contexts of miRNA loci.
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Affiliation(s)
- Soomin Son
- Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea
- School of Biological Sciences, Seoul National University, Seoul 08826, Korea
| | - Baekgyu Kim
- Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea
- School of Biological Sciences, Seoul National University, Seoul 08826, Korea
| | - Jihye Yang
- Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea
- School of Biological Sciences, Seoul National University, Seoul 08826, Korea
| | - V Narry Kim
- Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea;
- School of Biological Sciences, Seoul National University, Seoul 08826, Korea
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5
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de Rooij LA, Mastebroek DJ, ten Voorde N, van der Wall E, van Diest PJ, Moelans CB. The microRNA Lifecycle in Health and Cancer. Cancers (Basel) 2022; 14:cancers14235748. [PMID: 36497229 PMCID: PMC9736740 DOI: 10.3390/cancers14235748] [Citation(s) in RCA: 23] [Impact Index Per Article: 7.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2022] [Revised: 11/19/2022] [Accepted: 11/21/2022] [Indexed: 11/24/2022] Open
Abstract
MicroRNAs (miRNAs) are small non-coding RNAs of ~22 nucleotides that regulate gene expression at the post-transcriptional level. They can bind to around 60% of all protein-coding genes with an average of 200 targets per miRNA, indicating their important function within physiological and pathological cellular processes. miRNAs can be quickly produced in high amounts through canonical and non-canonical pathways that involve a multitude of steps and proteins. In cancer, miRNA biogenesis, availability and regulation of target expression can be altered to promote tumour progression. This can be due to genetic causes, such as single nucleotide polymorphisms, epigenetic changes, differences in host gene expression, or chromosomal remodelling. Alternatively, post-transcriptional changes in miRNA stability, and defective or absent components and mediators of the miRNA-induced silencing complex can lead to altered miRNA function. This review provides an overview of the current knowledge on the lifecycle of miRNAs in health and cancer. Understanding miRNA function and regulation is fundamental prior to potential future application of miRNAs as cancer biomarkers.
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Affiliation(s)
- Laura Adriana de Rooij
- Department of Pathology, University Medical Center Utrecht, Utrecht University, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
- Correspondence: ; Tel.: +31-887-556-557
| | - Dirk Jan Mastebroek
- Department of Pathology, University Medical Center Utrecht, Utrecht University, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
| | - Nicky ten Voorde
- Department of Pathology, University Medical Center Utrecht, Utrecht University, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
| | - Elsken van der Wall
- Department of Medical Oncology, University Medical Center Utrecht, Utrecht University, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
| | - Paul Joannes van Diest
- Department of Pathology, University Medical Center Utrecht, Utrecht University, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
| | - Cathy Beatrice Moelans
- Department of Pathology, University Medical Center Utrecht, Utrecht University, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
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HYL1-CLEAVAGE SUBTILASE 1 (HCS1) suppresses miRNA biogenesis in response to light-to-dark transition. Proc Natl Acad Sci U S A 2022; 119:2116757119. [PMID: 35121664 PMCID: PMC8833217 DOI: 10.1073/pnas.2116757119] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/16/2021] [Indexed: 01/02/2023] Open
Abstract
HYPONASTIC LEAVES 1 (HYL1)-CLEAVAGE SUBTILASE 1 (HCS1) is a novel negative regulator of microRNA (miRNA) biogenesis that degrades HYL1 in the cytoplasm. Furthermore, cytoplasm CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) E3 ligase inhibit HCS1-mediated HYL1 degradation. The COP1-HYL1-HCS1 network may integrate two essential cellular pathways: the miRNA-biogenetic pathway and light signaling pathway. Our finding suggests a regulatory pathway in the miRNA-biogenetic system. The core plant microprocessor consists of DICER-LIKE 1 (DCL1), SERRATE (SE), and HYPONASTIC LEAVES 1 (HYL1) and plays a pivotal role in microRNA (miRNA) biogenesis. However, the proteolytic regulation of each component remains elusive. Here, we show that HYL1-CLEAVAGE SUBTILASE 1 (HCS1) is a cytoplasmic protease for HYL1-destabilization. HCS1-excessiveness reduces HYL1 that disrupts miRNA biogenesis, while HCS1-deficiency accumulates HYL1. Consistently, we identified the HYL1K154A mutant that is insensitive to the proteolytic activity of HCS1, confirming the importance of HCS1 in HYL1 proteostasis. Moreover, HCS1-activity is regulated by light/dark transition. Under light, cytoplasmic CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) E3 ligase suppresses HCS1-activity. COP1 sterically inhibits HCS1 by obstructing HYL1 access into the catalytic sites of HCS1. In contrast, darkness unshackles HCS1-activity for HYL1-destabilization due to nuclear COP1 relocation. Overall, the COP1-HYL1-HCS1 network may integrate two essential cellular pathways: the miRNA-biogenetic pathway and light signaling pathway.
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7
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Hadj-Moussa H, Hawkins LJ, Storey KB. Role of MicroRNAs in Extreme Animal Survival Strategies. Methods Mol Biol 2022; 2257:311-347. [PMID: 34432286 DOI: 10.1007/978-1-0716-1170-8_16] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
The critical role microRNAs play in modulating global functions is emerging, both in the maintenance of homeostatic mechanisms and in the adaptation to diverse environmental stresses. When stressed, cells must divert metabolic requirements toward immediate survival and eventual recovery and the unique features of miRNAs, such as their relatively ATP-inexpensive biogenesis costs, and the quick and reversible nature of their action, renders them excellent "master controllers" for rapid responses. Many animal survival strategies for dealing with extreme environmental pressures involve prolonged retreats into states of suspended animation to extend the time that they can survive on their limited internal fuel reserves until conditions improve. The ability to retreat into such hypometabolic states is only possible by coupling the global suppression of nonessential energy-expensive functions with an activation of prosurvival networks, a process in which miRNAs are now known to play a major role. In this chapter, we discuss the activation, expression, biogenesis, and unique attributes of miRNA regulation required to facilitate profound metabolic rate depression and implement stress-specific metabolic adaptations. We examine the role of miRNA in strategies of biochemical adaptation including mammalian hibernation, freeze tolerance, freeze avoidance, anoxia and hypoxia survival, estivation, and dehydration tolerance. By comparing these seemingly different adaptive programs in traditional and exotic animal models, we highlight both unique and conserved miRNA-meditated mechanisms for survival. Additional topics discussed include transcription factor networks, temperature dependent miRNA-targeting, and novel species-specific and stress-specific miRNAs.
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Affiliation(s)
| | - Liam J Hawkins
- Department of Biology, Carleton University, Ottawa, ON, Canada
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8
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Mirahmadi Y, Nabavi R, Taheri F, Samadian MM, Ghale-Noie ZN, Farjami M, Samadi-khouzani A, Yousefi M, Azhdari S, Salmaninejad A, Sahebkar A. MicroRNAs as Biomarkers for Early Diagnosis, Prognosis, and Therapeutic Targeting of Ovarian Cancer. JOURNAL OF ONCOLOGY 2021; 2021:3408937. [PMID: 34721577 PMCID: PMC8553480 DOI: 10.1155/2021/3408937] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/29/2021] [Accepted: 09/27/2021] [Indexed: 02/06/2023]
Abstract
Ovarian cancer is the major cause of gynecologic cancer-related mortality. Regardless of outstanding advances, which have been made for improving the prognosis, diagnosis, and treatment of ovarian cancer, the majority of the patients will die of the disease. Late-stage diagnosis and the occurrence of recurrent cancer after treatment are the most important causes of the high mortality rate observed in ovarian cancer patients. Unraveling the molecular mechanisms involved in the pathogenesis of ovarian cancer may help find new biomarkers and therapeutic targets for ovarian cancer. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression, mostly at the posttranscriptional stage, through binding to mRNA targets and inducing translational repression or degradation of target via the RNA-induced silencing complex. Over the last two decades, the role of miRNAs in the pathogenesis of various human cancers, including ovarian cancer, has been documented in multiple studies. Consequently, these small RNAs could be considered as reliable markers for prognosis and early diagnosis. Furthermore, given the function of miRNAs in various cellular pathways, including cell survival and differentiation, targeting miRNAs could be an interesting approach for the treatment of human cancers. Here, we review our current understanding of the most updated role of the important dysregulation of miRNAs and their roles in the progression and metastasis of ovarian cancer. Furthermore, we meticulously discuss the significance of miRNAs as prognostic and diagnostic markers. Lastly, we mention the opportunities and the efforts made for targeting ovarian cancer through inhibition and/or stimulation of the miRNAs.
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Affiliation(s)
- Yegane Mirahmadi
- Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
- Medical Genetics Research Centre, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | | | - Fourough Taheri
- Department of Biology, Faculty of Basic Sciences, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
| | - Mohammad Mahdi Samadian
- Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Zari Naderi Ghale-Noie
- Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
- Medical Genetics Research Centre, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Mahsa Farjami
- Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
- Medical Genetics Research Centre, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Abbas Samadi-khouzani
- Department of Biology, Faculty of Basic Sciences, Shahrekord Branch, Islamic Azad University, Shahrekord, Iran
| | - Meysam Yousefi
- Department of Medical Genetics, Faculty of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Sara Azhdari
- Department of Anatomy and Embryology, School of Medicine, Bam University of Medical Sciences, Bam, Iran
| | - Arash Salmaninejad
- Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
- Medical Genetics Research Centre, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics, Faculty of Medicine, Guilan University of Medical Sciences, Guilan, Iran
| | - Amirhossein Sahebkar
- Applied Biomedical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Biotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran
- School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
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Lett KE, Logan MK, McLaurin DM, Hebert MD. Coilin enhances phosphorylation and stability of DGCR8 and promotes miRNA biogenesis. Mol Biol Cell 2021; 32:br4. [PMID: 34319763 PMCID: PMC8684749 DOI: 10.1091/mbc.e21-05-0225] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2021] [Revised: 07/15/2021] [Accepted: 07/20/2021] [Indexed: 11/11/2022] Open
Abstract
MicroRNAs (miRNAs) are ∼22 nt small noncoding RNAs that control gene expression at the posttranscriptional level through translational inhibition and destabilization of their target mRNAs. The biogenesis of miRNAs involves a series of processing steps beginning with cropping of the primary miRNA transcript by the Microprocessor complex, which is composed of Drosha and DGCR8. Here we report a novel regulatory interaction between the Microprocessor components and coilin, the Cajal body (CB) marker protein. Coilin knockdown causes alterations in the level of primary and mature miRNAs, let-7a and miR-34a, and their miRNA targets, HMGA2 and Notch1, respectively. We also found that coilin knockdown affects the levels of DGCR8 and Drosha in cells with (HeLa) and without (WI-38) CBs. To further explore the role of coilin in miRNA biogenesis, we conducted a series of coimmunoprecipitation experiments using coilin and DGCR8 constructs, which revealed that coilin and DGCR8 can form a complex. Additionally, our results indicate that phosphorylation of DGCR8, which has been shown to increase protein stability, is impacted by coilin knockdown. Collectively, our results implicate coilin as a member of the regulatory network governing miRNA biogenesis.
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Affiliation(s)
- Katheryn E. Lett
- Department of Cell and Molecular Biology, University of Mississippi Medical Center, Jackson, MS 39216-4505
| | - Madelyn K. Logan
- Department of Cell and Molecular Biology, University of Mississippi Medical Center, Jackson, MS 39216-4505
| | - Douglas M. McLaurin
- Department of Cell and Molecular Biology, University of Mississippi Medical Center, Jackson, MS 39216-4505
| | - Michael D. Hebert
- Department of Cell and Molecular Biology, University of Mississippi Medical Center, Jackson, MS 39216-4505
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10
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Tabatabaeian H, Lim SK, Chu T, Seah SH, Lim YP. WBP2 inhibits microRNA biogenesis via interaction with the microprocessor complex. Life Sci Alliance 2021; 4:4/7/e202101038. [PMID: 34117091 PMCID: PMC8200299 DOI: 10.26508/lsa.202101038] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2021] [Revised: 05/26/2021] [Accepted: 05/28/2021] [Indexed: 12/18/2022] Open
Abstract
WBP2 protein blocks the microRNA biogenesis via physical interactions with the microprocessor complex, and reverts the tumor-suppressive role of DGCR8. WBP2 is an emerging oncoprotein with diverse functions in breast tumorigenesis via regulating Wnt, epidermal growth factor receptor, estrogen receptor, and Hippo. Recently, evidence shows that WBP2 is tightly regulated by the components of the miRNA biogenesis machinery such as DGCR8 and Dicer via producing both WBP2’s 3′UTR and coding DNA sequence-targeting miRNAs. This led us to hypothesize that WBP2 could provide a feedback loop to the biogenesis of its key upstream regulators by regulating the microprocessor complex activity. Indeed, WBP2 suppressed microprocessor activity by blocking the processing of pri-miRNAs to pre-miRNAs. WBP2 negatively regulated the assembly of the microprocessor complex via physical interactions with its components. Meta-analyses suggest that microprocessor complex components, in particular DGCR8, DDX5, and DEAD-Box Helicase17 (DDX17), have tumor-suppressive properties. 2D and 3D in vitro proliferation assays revealed that WBP2 blocked the tumor-suppressive properties of DGCR8, a key component of the microprocessor complex. In conclusion, WBP2 is a novel regulator of miRNA biogenesis that is a known dysregulated pathway in breast tumorigenesis. The reregulation of miRNA biogenesis machinery via targeting WBP2 protein may have implications in breast cancer therapy.
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Affiliation(s)
- Hossein Tabatabaeian
- Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.,Cancer Science Institute of Singapore, National University of Singapore, Singapore
| | - Shen Kiat Lim
- Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.,School of Biological Sciences, Nanyang Technological University, Singapore
| | - Tinghine Chu
- Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.,National University of Singapore Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore
| | - Sock Hong Seah
- Mechanobiology Institute, National University of Singapore, Singapore
| | - Yoon Pin Lim
- Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore .,National University of Singapore Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore.,National University Cancer Institute, Singapore
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11
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Lee J, Lee M, Lee K. Trans-acting regulators of ribonuclease activity. J Microbiol 2021; 59:341-359. [PMID: 33779951 DOI: 10.1007/s12275-021-0650-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2020] [Revised: 12/28/2020] [Accepted: 12/28/2020] [Indexed: 12/16/2022]
Abstract
RNA metabolism needs to be tightly regulated in response to changes in cellular physiology. Ribonucleases (RNases) play an essential role in almost all aspects of RNA metabolism, including processing, degradation, and recycling of RNA molecules. Thus, living systems have evolved to regulate RNase activity at multiple levels, including transcription, post-transcription, post-translation, and cellular localization. In addition, various trans-acting regulators of RNase activity have been discovered in recent years. This review focuses on the physiological roles and underlying mechanisms of trans-acting regulators of RNase activity.
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Affiliation(s)
- Jaejin Lee
- Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea
| | - Minho Lee
- Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea.
| | - Kangseok Lee
- Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea.
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12
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Yang Y, Wang H, Li W, Wang X, Wei S, Liu Y, Xu Y. Prediction and analysis of multiple protein lysine modified sites based on conditional wasserstein generative adversarial networks. BMC Bioinformatics 2021; 22:171. [PMID: 33789579 PMCID: PMC8010967 DOI: 10.1186/s12859-021-04101-y] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2020] [Accepted: 03/23/2021] [Indexed: 01/05/2023] Open
Abstract
BACKGROUND Protein post-translational modification (PTM) is a key issue to investigate the mechanism of protein's function. With the rapid development of proteomics technology, a large amount of protein sequence data has been generated, which highlights the importance of the in-depth study and analysis of PTMs in proteins. METHOD We proposed a new multi-classification machine learning pipeline MultiLyGAN to identity seven types of lysine modified sites. Using eight different sequential and five structural construction methods, 1497 valid features were remained after the filtering by Pearson correlation coefficient. To solve the data imbalance problem, Conditional Generative Adversarial Network (CGAN) and Conditional Wasserstein Generative Adversarial Network (CWGAN), two influential deep generative methods were leveraged and compared to generate new samples for the types with fewer samples. Finally, random forest algorithm was utilized to predict seven categories. RESULTS In the tenfold cross-validation, accuracy (Acc) and Matthews correlation coefficient (MCC) were 0.8589 and 0.8376, respectively. In the independent test, Acc and MCC were 0.8549 and 0.8330, respectively. The results indicated that CWGAN better solved the existing data imbalance and stabilized the training error. Alternatively, an accumulated feature importance analysis reported that CKSAAP, PWM and structural features were the three most important feature-encoding schemes. MultiLyGAN can be found at https://github.com/Lab-Xu/MultiLyGAN . CONCLUSIONS The CWGAN greatly improved the predictive performance in all experiments. Features derived from CKSAAP, PWM and structure schemes are the most informative and had the greatest contribution to the prediction of PTM.
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Affiliation(s)
- Yingxi Yang
- Department of Information and Computer Science, University of Science and Technology Beijing, Beijing, 100083, China
| | - Hui Wang
- Institute of Computing Technology, Chinese Academy of Sciences, Beijing, 100080, China
| | - Wen Li
- Department of Information and Computer Science, University of Science and Technology Beijing, Beijing, 100083, China
| | - Xiaobo Wang
- Department of Information and Computer Science, University of Science and Technology Beijing, Beijing, 100083, China
| | - Shizhao Wei
- No. 15 Research Institute, China Electronics Technology Group Corporation, Beijing, 100083, China
| | - Yulong Liu
- No. 15 Research Institute, China Electronics Technology Group Corporation, Beijing, 100083, China
| | - Yan Xu
- Department of Information and Computer Science, University of Science and Technology Beijing, Beijing, 100083, China.
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13
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Lee J, Lee M, Lee K. Trans-acting regulators of ribonuclease activity. J Microbiol 2021:10.1007/s12275-021-0650-3. [PMID: 33565052 DOI: 10.1007/s12275-021-0650-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2020] [Revised: 12/28/2020] [Accepted: 12/28/2020] [Indexed: 11/29/2022]
Abstract
RNA metabolism needs to be tightly regulated in response to changes in cellular physiology. Ribonucleases (RNases) play an essential role in almost all aspects of RNA metabolism, including processing, degradation, and recycling of RNA molecules. Thus, living systems have evolved to regulate RNase activity at multiple levels, including transcription, post-transcription, post-translation, and cellular localization. In addition, various trans-acting regulators of RNase activity have been discovered in recent years. This review focuses on the physiological roles and underlying mechanisms of trans-acting regulators of RNase activity.
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Affiliation(s)
- Jaejin Lee
- Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea
| | - Minho Lee
- Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea.
| | - Kangseok Lee
- Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea.
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14
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Fofanov MV, Prokopov DY, Kuhl H, Schartl M, Trifonov VA. Evolution of MicroRNA Biogenesis Genes in the Sterlet ( Acipenser ruthenus) and Other Polyploid Vertebrates. Int J Mol Sci 2020; 21:E9562. [PMID: 33334059 PMCID: PMC7765534 DOI: 10.3390/ijms21249562] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2020] [Revised: 12/09/2020] [Accepted: 12/14/2020] [Indexed: 01/14/2023] Open
Abstract
MicroRNAs play a crucial role in eukaryotic gene regulation. For a long time, only little was known about microRNA-based gene regulatory mechanisms in polyploid animal genomes due to difficulties of polyploid genome assembly. However, in recent years, several polyploid genomes of fish, amphibian, and even invertebrate species have been sequenced and assembled. Here we investigated several key microRNA-associated genes in the recently sequenced sterlet (Acipenser ruthenus) genome, whose lineage has undergone a whole genome duplication around 180 MYA. We show that two paralogs of drosha, dgcr8, xpo1, and xpo5 as well as most ago genes have been retained after the acipenserid-specific whole genome duplication, while ago1 and ago3 genes have lost one paralog. While most diploid vertebrates possess only a single copy of dicer1, we strikingly found four paralogs of this gene in the sterlet genome, derived from a tandem segmental duplication that occurred prior to the last whole genome duplication. ago1,3,4 and exportins1,5 look to be prone to additional segment duplications producing up to four-five paralog copies in ray-finned fishes. We demonstrate for the first time exon microsatellite amplification in the acipenserid drosha2 gene, resulting in a highly variable protein product, which may indicate sub- or neofunctionalization. Paralogous copies of most microRNA metabolism genes exhibit different expression profiles in various tissues and remain functional despite the rediploidization process. Subfunctionalization of microRNA processing gene paralogs may be beneficial for different pathways of microRNA metabolism. Genetic variability of microRNA processing genes may represent a substrate for natural selection, and, by increasing genetic plasticity, could facilitate adaptations to changing environments.
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Affiliation(s)
- Mikhail V. Fofanov
- Institute of Molecular and Cellular Biology SB RAS, Lavrentiev Ave. 8/2, 630090 Novosibirsk, Russia;
- Department of Natural Sciences, Novosibirsk State University, Pirogova 2, 630090 Novosibirsk, Russia
| | - Dmitry Yu. Prokopov
- Institute of Molecular and Cellular Biology SB RAS, Lavrentiev Ave. 8/2, 630090 Novosibirsk, Russia;
| | - Heiner Kuhl
- Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Müggelseedamm 301 and 310, 12587 Berlin, Germany;
| | - Manfred Schartl
- Developmental Biochemistry, Biocenter, University of Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany;
- Xiphophorus Genetic Stock Center, Texas State University, 601 University Drive, 419 Centennial Hall, San Marcos, TX 78666-4616, USA
| | - Vladimir A. Trifonov
- Institute of Molecular and Cellular Biology SB RAS, Lavrentiev Ave. 8/2, 630090 Novosibirsk, Russia;
- Department of Natural Sciences, Novosibirsk State University, Pirogova 2, 630090 Novosibirsk, Russia
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15
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Nowak K, Morończyk J, Wójcik A, Gaj MD. AGL15 Controls the Embryogenic Reprogramming of Somatic Cells in Arabidopsis through the Histone Acetylation-Mediated Repression of the miRNA Biogenesis Genes. Int J Mol Sci 2020; 21:ijms21186733. [PMID: 32937992 PMCID: PMC7554740 DOI: 10.3390/ijms21186733] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2020] [Revised: 09/09/2020] [Accepted: 09/11/2020] [Indexed: 12/24/2022] Open
Abstract
The embryogenic transition of somatic cells requires an extensive reprogramming of the cell transcriptome. Relevantly, the extensive modulation of the genes that have a regulatory function, in particular the genes encoding the transcription factors (TFs) and miRNAs, have been indicated as controlling somatic embryogenesis (SE) that is induced in vitro in the somatic cells of plants. Identifying the regulatory relationships between the TFs and miRNAs during SE induction is of central importance for understanding the complex regulatory interplay that fine-tunes a cell transcriptome during the embryogenic transition. Hence, here, we analysed the regulatory relationships between AGL15 (AGAMOUS-LIKE 15) TF and miR156 in an embryogenic culture of Arabidopsis. Both AGL15 and miR156 control SE induction and AGL15 has been reported to target the MIR156 genes in planta. The results showed that AGL15 contributes to the regulation of miR156 in an embryogenic culture at two levels that involve the activation of the MIR156 transcription and the containment of the abundance of mature miR156 by repressing the miRNA biogenesis genes DCL1 (DICER-LIKE1), SERRATE and HEN1 (HUA-ENHANCER1). To repress the miRNA biogenesis genes AGL15 seems to co-operate with the TOPLESS co-repressors (TPL and TPR1-4), which are components of the SIN3/HDAC silencing complex. The impact of TSA (trichostatin A), an inhibitor of the HDAC histone deacetylases, on the expression of the miRNA biogenesis genes together with the ChIP results implies that histone deacetylation is involved in the AGL15-mediated repression of miRNA processing. The results indicate that HDAC6 and HDAC19 histone deacetylases might co-operate with AGL15 in silencing the complex that controls the abundance of miR156 during embryogenic induction. This study provides new evidence about the histone acetylation-mediated control of the miRNA pathways during the embryogenic reprogramming of plant somatic cells and the essential role of AGL15 in this regulatory mechanism.
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16
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Soleimani S, Valizadeh Arshad Z, Moradi S, Ahmadi A, Davarpanah SJ, Azimzadeh Jamalkandi S. Small regulatory noncoding RNAs in Drosophila melanogaster: biogenesis and biological functions. Brief Funct Genomics 2020; 19:309-323. [PMID: 32219422 DOI: 10.1093/bfgp/elaa005] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2019] [Revised: 02/15/2020] [Accepted: 02/19/2020] [Indexed: 02/06/2023] Open
Abstract
RNA interference (RNAi) is an important phenomenon that has diverse genetic regulatory functions at the pre- and posttranscriptional levels. The major trigger for the RNAi pathway is double-stranded RNA (dsRNA). dsRNA is processed to generate various types of major small noncoding RNAs (ncRNAs) that include microRNAs (miRNAs), small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) in Drosophila melanogaster (D. melanogaster). Functionally, these small ncRNAs play critical roles in virtually all biological systems and developmental pathways. Identification and processing of dsRNAs and activation of RNAi machinery are the three major academic interests that surround RNAi research. Mechanistically, some of the important biological functions of RNAi are achieved through: (i) supporting genomic stability via degradation of foreign viral genomes; (ii) suppressing the movement of transposable elements and, most importantly, (iii) post-transcriptional regulation of gene expression by miRNAs that contribute to regulation of epigenetic modifications such as heterochromatin formation and genome imprinting. Here, we review various routes of small ncRNA biogenesis, as well as different RNAi-mediated pathways in D. melanogaster with a particular focus on signaling pathways. In addition, a critical discussion of the most relevant and latest findings that concern the significant contribution of small ncRNAs to the regulation of D. melanogaster physiology and pathophysiology is presented.
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17
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Casali P, Shen T, Xu Y, Qiu Z, Chupp DP, Im J, Xu Z, Zan H. Estrogen Reverses HDAC Inhibitor-Mediated Repression of Aicda and Class-Switching in Antibody and Autoantibody Responses by Downregulation of miR-26a. Front Immunol 2020; 11:491. [PMID: 32265934 PMCID: PMC7105609 DOI: 10.3389/fimmu.2020.00491] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2020] [Accepted: 03/03/2020] [Indexed: 12/24/2022] Open
Abstract
Estrogen contributes to females' strong antibody response to microbial vaccines and proneness to autoimmunity, particularly antibody-mediated systemic autoimmunity, in females. We have hypothesized that this is due to estrogen-mediated potentiation of class switch DNA recombination (CSR) and somatic hypermutation (SHM). As we have shown, estrogen boosts AID expression, which is critical for both CSR and SHM, through upregulation of HoxC4, which together with NF-κB critically mediates Aicda (AID gene) promoter activation. We contend here that additional regulation of Aicda expression by estrogen occurs through epigenetic mechanisms. As we have shown, histone deacetylase inhibitors (HDIs) short-chain fatty acid (SCFA) butyrate and propionate as well as the pharmacologic HDI valproic acid upregulate miRNAs that silence AID expression, thereby modulating specific antibody responses in C57BL/6 mice and autoantibody responses in lupus-prone MRL/Faslpr/lpr mice. Here, using constitutive knockout Esr1-/- mice and B cells as well as conditional knockout Aicdacre/creEsr1flox/flox mice and B cells, we showed that the HDI-mediated downregulation of Aicda expression as well as the maturation of antibody and autoantibody responses is reversed by estrogen and enhanced by deletion of ERα or E2 inhibition. Estrogen's reversion of HDI-mediated inhibition of Aicda and CSR in antibody and autoantibody responses occurred through downregulation of B cell miR-26a, which, as we showed, targets Aicda mRNA 3'UTR. miR-26a was significantly upregulated by HDIs. Accordingly, enforced expression of miR-26a reduced Aicda expression and CSR, while miR-26a-sponges (competitive inhibitors of miR-26a) increased Aicda expression and CSR. Thus, our findings show that estrogen reverses the HDI-mediated downregulation of AID expression and CSR through selective modulation of miR-26a. They also provide mechanistic insights into the immunomodulatory activity of this hormone and a proof-of-principle for using combined ER inhibitor-HDI as a potential therapeutic approach.
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Affiliation(s)
- Paolo Casali
- Department of Microbiology, Immunology & Molecular Genetics, University of Texas Long School of Medicine, UT Health San Antonio, San Antonio, TX, United States
| | | | | | | | | | | | | | - Hong Zan
- Department of Microbiology, Immunology & Molecular Genetics, University of Texas Long School of Medicine, UT Health San Antonio, San Antonio, TX, United States
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18
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DROSHA-Dependent miRNA and AIM2 Inflammasome Activation in Idiopathic Pulmonary Fibrosis. Int J Mol Sci 2020; 21:ijms21051668. [PMID: 32121297 PMCID: PMC7084700 DOI: 10.3390/ijms21051668] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2020] [Revised: 02/23/2020] [Accepted: 02/27/2020] [Indexed: 12/11/2022] Open
Abstract
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease. Chronic lung inflammation is linked to the pathogenesis of IPF. DROSHA, a class 2 ribonuclease III enzyme, has an important role in the biogenesis of microRNA (miRNA). The function of miRNAs has been identified in the regulation of the target gene or protein related to inflammatory responses via degradation of mRNA or inhibition of translation. The absent-in-melanoma-2 (AIM2) inflammasome is critical for inflammatory responses against cytosolic double stranded DNA (dsDNA) from pathogen-associated molecular patterns (PAMPs) and self-DNA from danger-associated molecular patterns (DAMPs). The AIM2 inflammasome senses double strand DNA (dsDNA) and interacts with the adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which recruits pro-caspase-1 and regulates the maturation and secretion of interleukin (IL)-1β and IL-18. A recent study showed that inflammasome activation contributes to lung inflammation and fibrogenesis during IPF. In the current review, we discuss recent advances in our understanding of the DROSHA-miRNA-AIM2 inflammasome axis in the pathogenesis of IPF.
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19
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Spadotto V, Giambruno R, Massignani E, Mihailovich M, Maniaci M, Patuzzo F, Ghini F, Nicassio F, Bonaldi T. PRMT1-mediated methylation of the microprocessor-associated proteins regulates microRNA biogenesis. Nucleic Acids Res 2020; 48:96-115. [PMID: 31777917 PMCID: PMC6943135 DOI: 10.1093/nar/gkz1051] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2018] [Revised: 10/04/2019] [Accepted: 11/22/2019] [Indexed: 12/17/2022] Open
Abstract
MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Microprocessor and its associated proteins. Through high resolution mass spectrometry (MS)- proteomics we discovered that this complex is extensively methylated, with 84 methylated sites associated to 19 out of its 24 subunits. The majority of the modifications occurs on arginine (R) residues (61), leading to 81 methylation events, while 30 lysine (K)-methylation events occurs on 23 sites of the complex. Interestingly, both depletion and pharmacological inhibition of the Type-I Protein Arginine Methyltransferases (PRMTs) lead to a widespread change in the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the Microprocessor subunit ILF3 is linked to its diminished binding to the pri-miRNAs miR-15a/16, miR-17-92, miR-301a and miR-331. Our study uncovers a previously uncharacterized role of R-methylation in the regulation of miRNA biogenesis in mammalian cells.
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Affiliation(s)
- Valeria Spadotto
- Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy
| | - Roberto Giambruno
- Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy
| | - Enrico Massignani
- Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy
| | - Marija Mihailovich
- Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy
| | - Marianna Maniaci
- Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy
| | - Francesca Patuzzo
- Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy
| | - Francesco Ghini
- Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia, Milan, Italy
| | - Francesco Nicassio
- Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia, Milan, Italy
| | - Tiziana Bonaldi
- Department of Experimental Oncology, IEO, European Institute of Oncology IRCCS, Milan, Italy
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20
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Sanchez HN, Moroney JB, Gan H, Shen T, Im JL, Li T, Taylor JR, Zan H, Casali P. B cell-intrinsic epigenetic modulation of antibody responses by dietary fiber-derived short-chain fatty acids. Nat Commun 2020; 11:60. [PMID: 31896754 PMCID: PMC6940392 DOI: 10.1038/s41467-019-13603-6] [Citation(s) in RCA: 232] [Impact Index Per Article: 46.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2018] [Accepted: 11/12/2019] [Indexed: 02/06/2023] Open
Abstract
Short-chain fatty acids (SCFAs) butyrate and propionate are metabolites from dietary fiber's fermentation by gut microbiota that can affect differentiation or functions of T cells, macrophages and dendritic cells. We show here that at low doses these SCFAs directly impact B cell intrinsic functions to moderately enhance class-switch DNA recombination (CSR), while decreasing at higher doses over a broad physiological range, AID and Blimp1 expression, CSR, somatic hypermutation and plasma cell differentiation. In human and mouse B cells, butyrate and propionate decrease B cell Aicda and Prdm1 by upregulating select miRNAs that target Aicda and Prdm1 mRNA-3′UTRs through inhibition of histone deacetylation (HDAC) of those miRNA host genes. By acting as HDAC inhibitors, not as energy substrates or through GPR-engagement signaling in these B cell-intrinsic processes, these SCFAs impair intestinal and systemic T-dependent and T-independent antibody responses. Their epigenetic impact on B cells extends to inhibition of autoantibody production and autoimmunity in mouse lupus models. Dietary fiber-derived short-chain fatty acids (SCFA) act as histone deacetylase (HDAC) inhibitors on Tregs and innate immune cells, promoting immune tolerance by altering gene expression. Here the authors show that SCFA HDAC inhibitor activity impacts B cell differentiation, antibody responses and antibody-driven autoimmunity.
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Affiliation(s)
- Helia N Sanchez
- Department of Microbiology, Immunology & Molecular Genetics, University of Texas Long School of Medicine, UT Health Science Center, San Antonio, TX, 78229, USA
| | - Justin B Moroney
- Department of Microbiology, Immunology & Molecular Genetics, University of Texas Long School of Medicine, UT Health Science Center, San Antonio, TX, 78229, USA
| | - Huoqun Gan
- Department of Microbiology, Immunology & Molecular Genetics, University of Texas Long School of Medicine, UT Health Science Center, San Antonio, TX, 78229, USA.,Xiangya School of Medicine, Central South University, Changsha, Hunan, 410011, China
| | - Tian Shen
- Department of Microbiology, Immunology & Molecular Genetics, University of Texas Long School of Medicine, UT Health Science Center, San Antonio, TX, 78229, USA.,Xiangya School of Medicine, Central South University, Changsha, Hunan, 410011, China
| | - John L Im
- Department of Microbiology, Immunology & Molecular Genetics, University of Texas Long School of Medicine, UT Health Science Center, San Antonio, TX, 78229, USA
| | - Tianbao Li
- Department of Molecular Medicine, University of Texas Long School of Medicine, UT Health Science Center, San Antonio, TX, 78229, USA
| | - Julia R Taylor
- Department of Microbiology, Immunology & Molecular Genetics, University of Texas Long School of Medicine, UT Health Science Center, San Antonio, TX, 78229, USA
| | - Hong Zan
- Department of Microbiology, Immunology & Molecular Genetics, University of Texas Long School of Medicine, UT Health Science Center, San Antonio, TX, 78229, USA.
| | - Paolo Casali
- Department of Microbiology, Immunology & Molecular Genetics, University of Texas Long School of Medicine, UT Health Science Center, San Antonio, TX, 78229, USA.
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21
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Fabris G, Dumortier O, Pisani DF, Gautier N, Van Obberghen E. Amino acid-induced regulation of hepatocyte growth: possible role of Drosha. Cell Death Dis 2019; 10:566. [PMID: 31332188 PMCID: PMC6646398 DOI: 10.1038/s41419-019-1779-7] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2019] [Revised: 05/23/2019] [Accepted: 06/21/2019] [Indexed: 12/14/2022]
Abstract
In an adult healthy liver, hepatocytes are in a quiescent stage unless a physical injury, such as ablation, or a toxic attack occur. Indeed, to maintain their crucial organismal homeostatic role, the damaged or remaining hepatocytes will start proliferating to restore their functional mass. One of the limiting conditions for cell proliferation is amino-acid availability, necessary both for the synthesis of proteins important for cell growth and division, and for the activation of the mTOR pathway, known for its considerable role in the regulation of cell proliferation. The overarching aim of our present work was to investigate the role of amino acids in the regulation of the switch between quiescence and growth of adult hepatocytes. To do so we used non-confluent primary adult rat hepatocytes as a model of partially ablated liver. We discovered that the absence of amino acids induces in primary rat hepatocytes the entrance in a quiescence state together with an increase in Drosha protein, which does not involve the mTOR pathway. Conversely, Drosha knockdown allows the hepatocytes, quiescent after amino-acid deprivation, to proliferate again. Further, hepatocyte proliferation appears to be independent of miRNAs, the canonical downstream partners of Drosha. Taken together, our observations reveal an intriguing non-canonical action of Drosha in the control of growth regulation of adult hepatocytes responding to a nutritional strain, and they may help to design novel preventive and/or therapeutic approaches for hepatic failure.
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Affiliation(s)
- Gaia Fabris
- Université Côte d'Azur, Inserm, CNRS, IRCAN, Nice, France.,Université Côte d'Azur, CNRS, LP2M, Nice, France
| | | | | | - Nadine Gautier
- Université Côte d'Azur, Inserm, CNRS, IRCAN, Nice, France.,Université Côte d'Azur, CNRS, Inserm, iBV, Nice, France
| | - Emmanuel Van Obberghen
- Université Côte d'Azur, CHU, Inserm, CNRS, IRCAN, Nice, France. .,Université Côte d'Azur, CHU, CNRS, LP2M, Nice, France.
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22
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Huang L, Liu X. microRNA-370 Promotes Cell Growth by Targeting WNK2 in Breast Cancer. DNA Cell Biol 2019; 38:501-509. [PMID: 31009242 DOI: 10.1089/dna.2018.4602] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Affiliation(s)
- Li Huang
- Central Hospital of Zibo, Zhangdian District, Zibo, Shandong, China
| | - Xiangyu Liu
- Central Hospital of Zibo, Zhangdian District, Zibo, Shandong, China
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23
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Nguyen HM, Nguyen TD, Nguyen TL, Nguyen TA. Orientation of Human Microprocessor on Primary MicroRNAs. Biochemistry 2018; 58:189-198. [DOI: 10.1021/acs.biochem.8b00944] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Affiliation(s)
- Huong Minh Nguyen
- Laboratory of Molecular Microbiology, Institute of Biotechnology, Vietnam Academy of Science and Technology, Hanoi, Vietnam
| | - Trung Duc Nguyen
- Division of Life Science, Hong Kong University of Science & Technology, Hong Kong, China
| | - Thuy Linh Nguyen
- Division of Life Science, Hong Kong University of Science & Technology, Hong Kong, China
| | - Tuan Anh Nguyen
- Division of Life Science, Hong Kong University of Science & Technology, Hong Kong, China
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24
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Hadj-Moussa H, Storey KB. Micromanaging freeze tolerance: the biogenesis and regulation of neuroprotective microRNAs in frozen brains. Cell Mol Life Sci 2018; 75:3635-3647. [PMID: 29681008 PMCID: PMC11105625 DOI: 10.1007/s00018-018-2821-0] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2017] [Revised: 04/08/2018] [Accepted: 04/17/2018] [Indexed: 12/21/2022]
Abstract
When temperatures plummet below 0 °C, wood frogs (Rana sylvatica) can endure the freezing of up to ~ 65% of their body water in extracellular ice masses, displaying no measurable brain activity, no breathing, no movement, and a flat-lined heart. To aid survival, frogs retreat into a state of suspended animation characterized by global suppression of metabolic functions and reprioritization of energy usage to essential survival processes that is elicited, in part, by the regulatory controls of microRNAs. The present study is the first to investigate miRNA biogenesis and regulation in the brain of a freeze tolerant vertebrate. Indeed, proper brain function and adaptations to environmental stimuli play a crucial role in coordinating stress responses. Immunoblotting of miRNA biogenesis factors illustrated an overall reduction in the majority of these processing proteins suggesting a potential suppression of miRNA maturation over the freeze-thaw cycle. This was coupled with a large-scale RT-qPCR analysis of relative expression levels of 113 microRNA species in the brains of control, 24 h frozen, and 8 h thawed R. sylvatica. Of the 41 microRNAs differentially regulated during freezing and thawing, only two were significantly upregulated. Bioinformatic target enrichment of the downregulated miRNAs, performed at the low temperatures experienced during freezing and thawing, predicted their involvement in the potential activation of various neuroprotective processes such as synaptic signaling, intracellular signal transduction, and anoxia/ischemia injury protection. The predominantly downregulated microRNA fingerprint identified herein suggests a microRNA-mediated cryoprotective mechanism responsible for maintaining neuronal functions and facilitating successful whole brain freezing and thawing.
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Affiliation(s)
- Hanane Hadj-Moussa
- Department of Biology and Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada
| | - Kenneth B Storey
- Department of Biology and Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada.
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25
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Zhou L, Lim MYT, Kaur P, Saj A, Bortolamiol-Becet D, Gopal V, Tolwinski N, Tucker-Kellogg G, Okamura K. Importance of miRNA stability and alternative primary miRNA isoforms in gene regulation during Drosophila development. eLife 2018; 7:e38389. [PMID: 30024380 PMCID: PMC6066331 DOI: 10.7554/elife.38389] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2018] [Accepted: 07/04/2018] [Indexed: 12/19/2022] Open
Abstract
Mature microRNAs (miRNAs) are processed from primary transcripts (pri-miRNAs), and their expression is controlled at transcriptional and post-transcriptional levels. However, how regulation at multiple levels achieves precise control remains elusive. Using published and new datasets, we profile a time course of mature and pri-miRNAs in Drosophila embryos and reveal the dynamics of miRNA production and degradation as well as dynamic changes in pri-miRNA isoform selection. We found that 5' nucleotides influence stability of mature miRNAs. Furthermore, distinct half-lives of miRNAs from the mir-309 cluster shape their temporal expression patterns, and the importance of rapid degradation of the miRNAs in gene regulation is detected as distinct evolutionary signatures at the target sites in the transcriptome. Finally, we show that rapid degradation of miR-3/-309 may be important for regulation of the planar cell polarity pathway component Vang. Altogether, the results suggest that complex mechanisms regulate miRNA expression to support normal development.
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Affiliation(s)
- Li Zhou
- Temasek Life Sciences LaboratorySingaporeSingapore
- Department of Biological Sciences, Faculty of ScienceNational University of SingaporeSingaporeSingapore
| | - Mandy Yu Theng Lim
- Temasek Life Sciences LaboratorySingaporeSingapore
- School of Biological SciencesNanyang Technological UniversitySingaporeSingapore
| | - Prameet Kaur
- Division of ScienceYale-NUS CollegeSingaporeSingapore
| | - Abil Saj
- Cancer Therapeutics and Stratified OncologyGenome Institute of SingaporeSingaporeSingapore
| | | | - Vikneswaran Gopal
- Department of Statistics and Applied Probability, Faculty of ScienceNational University of SingaporeSingaporeSingapore
| | - Nicholas Tolwinski
- Department of Biological Sciences, Faculty of ScienceNational University of SingaporeSingaporeSingapore
- Division of ScienceYale-NUS CollegeSingaporeSingapore
| | - Greg Tucker-Kellogg
- Department of Biological Sciences, Faculty of ScienceNational University of SingaporeSingaporeSingapore
| | - Katsutomo Okamura
- Temasek Life Sciences LaboratorySingaporeSingapore
- School of Biological SciencesNanyang Technological UniversitySingaporeSingapore
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26
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Creugny A, Fender A, Pfeffer S. Regulation of primary microRNA processing. FEBS Lett 2018; 592:1980-1996. [PMID: 29683487 DOI: 10.1002/1873-3468.13067] [Citation(s) in RCA: 50] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2018] [Revised: 04/12/2018] [Accepted: 04/16/2018] [Indexed: 12/28/2022]
Abstract
MicroRNAs (miRNAs) are evolutionarily conserved small regulatory RNAs that participate in the adjustment of many, if not all, fundamental biological processes. Molecular mechanisms involved in miRNA biogenesis and mode of action have been elucidated in the past two decades. Similar to many cellular pathways, miRNA processing and function can be globally or specifically regulated at several levels and by numerous proteins and RNAs. Given their role as fine-tuning molecules, it is essential for miRNA expression to be tightly regulated in order to maintain cellular homeostasis. Here, we review our current knowledge of the first step of their maturation occurring in the nucleus and how it can be specifically and dynamically modulated.
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Affiliation(s)
- Antoine Creugny
- Architecture and Reactivity of RNA, Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, France
| | - Aurélie Fender
- Architecture and Reactivity of RNA, Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, France
| | - Sébastien Pfeffer
- Architecture and Reactivity of RNA, Institut de Biologie Moléculaire et Cellulaire du CNRS, Université de Strasbourg, France
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27
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Ko CY, Lin CH, Chuang JY, Chang WC, Hsu TI. MDM2 Degrades Deacetylated Nucleolin Through Ubiquitination to Promote Glioma Stem-Like Cell Enrichment for Chemotherapeutic Resistance. Mol Neurobiol 2018; 55:3211-3223. [PMID: 28478507 DOI: 10.1007/s12035-017-0569-4] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2017] [Accepted: 04/20/2017] [Indexed: 12/20/2022]
Abstract
Glioblastoma multiforme (GBM) is the most fatal of all brain cancers, and the standard care protocol for GBM patients is surgical tumor resection followed by radiotherapy and temozolomide (TMZ)-mediated chemotherapy. However, tumor recurrence frequently occurs, and recurrent GBM exhibits more malignancy and less sensitivity in response to chemotherapy. The malignancy and drug resistance primarily reflect the small population of glioma stem-like cells (GSC). Therefore, understanding the mechanism that controls GSC enrichment is important to benefit the prognosis of GBM patients. Nucleolin (NCL), which is responsible for ribosome biogenesis and RNA maturation, is overexpressed in gliomas. However, the role of NCL in GSC development and drug resistance is still unclear. In this study, we demonstrate that NCL attenuated GSC enrichment to enhance the sensitivity of GBM cells in response to TMZ. In GSC enrichment, NCL was significantly reduced at the protein level as a result of decreased protein stability. In particular, the inhibition of HDAC activity by suberoylanilide hydroxamic acid rescued NCL acetylation accompanied by the loss of mouse double minute 2 homolog (MDM2)-mediated ubiquitination. In addition, we found that NCL ubiquitination resulted from the activation of STAT3- and JNK-mediated signaling in GSC. Moreover, NCL inhibited the formation of stem-like spheres by attenuating the expression of Sox2, Oct4, and Bmi1. Furthermore, NCL sensitized the response of GBM cells to TMZ. Based on these findings, NCL expression is a potential indicator to predict chemotherapeutic efficiency in GBM patients.
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MESH Headings
- Acetylation
- Brain Neoplasms/metabolism
- Brain Neoplasms/pathology
- Cell Line, Tumor
- Down-Regulation/genetics
- Drug Resistance, Neoplasm/drug effects
- Drug Resistance, Neoplasm/genetics
- Gene Expression Regulation, Neoplastic/drug effects
- Glioma/genetics
- Glioma/metabolism
- Glioma/pathology
- Histone Deacetylase Inhibitors/pharmacology
- Humans
- JNK Mitogen-Activated Protein Kinases/metabolism
- Models, Biological
- Neoplasm Recurrence, Local/genetics
- Neoplasm Recurrence, Local/pathology
- Neoplastic Stem Cells/metabolism
- Neoplastic Stem Cells/pathology
- Phosphoproteins/metabolism
- Phosphorylation/drug effects
- Proteolysis/drug effects
- Proto-Oncogene Proteins c-mdm2/metabolism
- RNA, Messenger/genetics
- RNA, Messenger/metabolism
- RNA-Binding Proteins/metabolism
- STAT3 Transcription Factor/metabolism
- Spheroids, Cellular/metabolism
- Spheroids, Cellular/pathology
- Temozolomide/pharmacology
- Ubiquitination
- Vorinostat/pharmacology
- Nucleolin
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Affiliation(s)
- Chiung-Yuan Ko
- The Ph.D. Program for Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University and National Health Research Institutes, Taipei, Taiwan
| | - Chao-Han Lin
- Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan
| | - Jian-Ying Chuang
- The Ph.D. Program for Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University and National Health Research Institutes, Taipei, Taiwan
- Comprehensive Cancer Center, College of Medicine, Taipei Medical University, Taipei, Taiwan
| | - Wen-Chang Chang
- The Ph.D. Program for Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University and National Health Research Institutes, Taipei, Taiwan
- Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan
- Comprehensive Cancer Center, College of Medicine, Taipei Medical University, Taipei, Taiwan
| | - Tsung-I Hsu
- The Ph.D. Program for Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University and National Health Research Institutes, Taipei, Taiwan.
- Comprehensive Cancer Center, College of Medicine, Taipei Medical University, Taipei, Taiwan.
- Center for Neurotrauma and Neuroregeneration, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
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28
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Abstract
DROSHA is the catalytic subunit of the Microprocessor complex, which initiates microRNA (miRNA) maturation in the nucleus by recognizing and cleaving hairpin precursors embedded in primary transcripts. However, accumulating evidence suggests that not all hairpin substrates of DROSHA are associated with the generation of functional small RNAs. By targeting those hairpins, DROSHA regulates diverse aspects of RNA metabolism across the transcriptome, serves as a line of defense against the expression of potentially deleterious elements, and permits cell fate determination and differentiation. DROSHA is also versatile in the way that it executes these noncanonical functions, occasionally depending on its RNA-binding activity rather than its catalytic activity. Herein, we discuss the functional and mechanistic diversity of DROSHA beyond the miRNA biogenesis pathway in light of recent findings.
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Affiliation(s)
- Dooyoung Lee
- a Department of Agricultural Biotechnology , Seoul National University , Seoul , Republic of Korea
| | - Chanseok Shin
- a Department of Agricultural Biotechnology , Seoul National University , Seoul , Republic of Korea.,b Research Institute of Agriculture and Life Sciences, and Plant Genomics and Breeding Institute , Seoul National University , Seoul , Republic of Korea
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29
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Lee D, Nam JW, Shin C. DROSHA targets its own transcript to modulate alternative splicing. RNA (NEW YORK, N.Y.) 2017; 23:1035-1047. [PMID: 28400409 PMCID: PMC5473138 DOI: 10.1261/rna.059808.116] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/27/2016] [Accepted: 04/06/2017] [Indexed: 05/23/2023]
Abstract
The nuclear RNase III enzyme DROSHA interacts with its cofactor DGCR8 to form the Microprocessor complex, which initiates microRNA (miRNA) maturation by cleaving hairpin structures embedded in primary transcripts. Apart from its central role in the biogenesis of miRNAs, DROSHA is also known to recognize and cleave miRNA-like hairpins in a subset of transcripts without apparent small RNA production. Here, we report that the human DROSHA transcript is one such noncanonical target of DROSHA. Mammalian DROSHA genes have evolved a conserved hairpin structure spanning a specific exon-intron junction, which serves as a substrate for the Microprocessor in human cells but not in murine cells. We show that it is this hairpin element that decides whether the overlapping exon is alternatively or constitutively spliced. We further demonstrate that DROSHA promotes skipping of the overlapping exon in human cells independently of its cleavage function. Our findings add to the expanding list of noncanonical DROSHA functions.
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Affiliation(s)
- Dooyoung Lee
- Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea
| | - Jin-Wu Nam
- Department of Life Science, Hanyang University, Seoul 04763, Republic of Korea
| | - Chanseok Shin
- Department of Agricultural Biotechnology, Seoul National University, Seoul 08826, Republic of Korea
- Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul 08826, Republic of Korea
- Plant Genomics and Breeding Institute, Seoul National University, Seoul 08826, Republic of Korea
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30
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Identification of a Specific miRNA Profile in HIV-Exposed Seronegative Individuals. J Acquir Immune Defic Syndr 2017; 73:11-9. [PMID: 27171739 DOI: 10.1097/qai.0000000000001070] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
OBJECTIVE MicroRNAs (miRNAs) are small noncoding RNAs involved in the posttranscriptional regulation of gene expression that play important roles in viral infections. Alterations of specific miRNAs are described in HIV infection, suggesting a role for miRNAs in pathogenesis of this disease. We verified whether a particular miRNA signature could be identified in natural resistance to HIV-1. METHODS Expression level of 84 miRNAs was analyzed by RT-qPCR in plasma and unstimulated peripheral blood mononuclear cell (PBMC) of 30 seronegative individuals repeatedly exposed to HIV-1 (HESN), 30 HIV seropositive subjects (HIV+), and 30 healthy controls (HC). Results were confirmed by individual RT-qPCR in in vitro HIV-1-infected PBMC and in their cell culture medium. Dicer and Drosha expression was analyzed in basal PBMC. RESULTS Whereas Dicer and Drosha expression was comparable in HESN, HIV+ and HC, several miRNAs were upregulated both in HESN and HIV+ compared with HC. Furthermore, miRNA-29a and miR-223 were upregulated in both unstimulated PBMC and plasma of HESN alone; their expression was reduced upon in vitro HIV-1 infection of HESN PBMC indicating that, upon infection, they are secreted in the extracellular milieu. These results were confirmed by individual qPCR. CONCLUSIONS Our studies demonstrate that HIV-1 exposure modifies miRNAs expression even in the absence of productive infection. Because those miRNAs that are specifically increased only in HESN have been known to reduce HIV-1 replication, their modulation could represent an important mechanism in resistance to HIV-1 infection.
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31
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Binding and inhibition of the ternary complex factor Elk-4/Sap1 by the adapter protein Dok-4. Biochem J 2017; 474:1509-1528. [PMID: 28275114 DOI: 10.1042/bcj20160832] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/05/2016] [Revised: 02/17/2017] [Accepted: 03/08/2017] [Indexed: 01/25/2023]
Abstract
The adapter protein Dok-4 (downstream of kinase-4) has been reported as both an activator and inhibitor of Erk and Elk-1, but lack of knowledge about the identity of its partner molecules has precluded any mechanistic insight into these seemingly conflicting properties. We report that Dok-4 interacts with the transactivation domain of Elk-4 through an atypical phosphotyrosine-binding domain-mediated interaction. Dok-4 possesses a nuclear export signal and can relocalize Elk-4 from nucleus to cytosol, whereas Elk-4 possesses two nuclear localization signals that restrict interaction with Dok-4. The Elk-4 protein, unlike Elk-1, is highly unstable in the presence of Dok-4, through both an interaction-dependent mechanism and a pleckstrin homology domain-dependent but interaction-independent mechanism. This is reversed by proteasome inhibition, depletion of endogenous Dok-4 or lysine-to-arginine mutation of putative Elk-4 ubiquitination sites. Finally, Elk-4 transactivation is potently inhibited by Dok-4 overexpression but enhanced by Dok-4 knockdown in MDCK renal tubular cells, which correlates with increased basal and EGF-induced expression of Egr-1, Fos and cylcinD1 mRNA, and cell proliferation despite reduced Erk activation. Thus, Dok-4 can target Elk-4 activity through multiple mechanisms, including binding of the transactivation domain, nuclear exclusion and protein destabilization, without a requirement for inhibition of Erk.
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Cytoplasmic Drosha Is Aberrant in Precancerous Lesions of Gastric Carcinoma and Its Loss Predicts Worse Outcome for Gastric Cancer Patients. Dig Dis Sci 2016; 61:1080-90. [PMID: 26694172 DOI: 10.1007/s10620-015-3986-0] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/20/2015] [Accepted: 11/30/2015] [Indexed: 01/13/2023]
Abstract
BACKGROUND The nuclear localization of Drosha is critical for its function as a microRNA maturation regulator. Dephosphorylation of Drosha at serine 300 and serine 302 disrupts its nuclear localization, and aberrant distribution of Drosha has been detected in some tumors. AIMS The purpose of the present study was to assess cytoplasmic/nuclear Drosha expression in gastric cancer carcinogenesis and progression. METHODS Drosha expression and its subcellular location was investigated by immunohistochemical staining of a set of tissue microarrays composed of normal adjacent tissues (374), chronic gastritis (137), precancerous lesions (94), and gastric adenocarcinoma (829) samples, and in gastric cancer cell lines with varying differentiation by immunofluorescence and western blot assay. RESULTS Gradual loss of cytoplasmic Drosha was accompanied by tumor progression in both gastric cancer tissues and cell lines, and was inversely associated with tumor volume (P = 0.002), tumor grade (P < 0.001), tumor stage (P = 0.018), and distant metastasis (P = 0.026). Aberrant high levels of cytoplasmic Drosha were apparent in intestinal metaplasia and dysplasia tissues. The levels of nuclear Drosha were sharply decreased in chronic gastritis and maintained through precancerous lesions to gastric cancer. High levels of cytoplasmic Drosha predicted longer survival (LR = 7.088, P = 0.008) in gastric cancer patients. CONCLUSIONS Our data provide novel insights into gastric cancer that cytoplasmic Drosha potentially plays a role in preventing carcinogenesis and tumor progression, and may be an independent predictor of patient outcome.
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33
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Lahiri DK, Maloney B, Bayon BL, Chopra N, White FA, Greig NH, Nurnberger JI. Transgenerational latent early-life associated regulation unites environment and genetics across generations. Epigenomics 2016; 8:373-87. [PMID: 26950428 DOI: 10.2217/epi.15.117] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023] Open
Abstract
The origin of idiopathic diseases is still poorly understood. The latent early-life associated regulation (LEARn) model unites environmental exposures and gene expression while providing a mechanistic underpinning for later-occurring disorders. We propose that this process can occur across generations via transgenerational LEARn (tLEARn). In tLEARn, each person is a 'unit' accumulating preclinical or subclinical 'hits' as in the original LEARn model. These changes can then be epigenomically passed along to offspring. Transgenerational accumulation of 'hits' determines a sporadic disease state. Few significant transgenerational hits would accompany conception or gestation of most people, but these may suffice to 'prime' someone to respond to later-life hits. Hits need not produce symptoms or microphenotypes to have a transgenerational effect. Testing tLEARn requires longitudinal approaches. A recently proposed longitudinal epigenome/envirome-wide association study would unite genetic sequence, epigenomic markers, environmental exposures, patient personal history taken at multiple time points and family history.
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Affiliation(s)
- Debomoy K Lahiri
- Department of Psychiatry, Stark Neurosciences Research Institute, Indiana University School of Medicine, 320 West 15th Street, Indianapolis, IN 46202, USA.,Department of Medical & Molecular Genetics, Indiana University School of Medicine, 320 West 15th Street, Indianapolis, IN 46202, USA
| | - Bryan Maloney
- Department of Psychiatry, Stark Neurosciences Research Institute, Indiana University School of Medicine, 320 West 15th Street, Indianapolis, IN 46202, USA
| | - Baindu L Bayon
- Department of Medical & Molecular Genetics, Indiana University School of Medicine, 320 West 15th Street, Indianapolis, IN 46202, USA
| | - Nipun Chopra
- Department of Psychiatry, Stark Neurosciences Research Institute, Indiana University School of Medicine, 320 West 15th Street, Indianapolis, IN 46202, USA
| | - Fletcher A White
- Department of Anesthesia, Stark Neurosciences Research Institute, Indiana University School of Medicine, 320 West 15th Street, Indianapolis, IN 46202, USA
| | - Nigel H Greig
- Translational Gerontology Branch, National Institute on Aging, NIH, Baltimore, MD 21224, USA
| | - John I Nurnberger
- Department of Psychiatry, Stark Neurosciences Research Institute, Indiana University School of Medicine, 320 West 15th Street, Indianapolis, IN 46202, USA.,Department of Medical & Molecular Genetics, Indiana University School of Medicine, 320 West 15th Street, Indianapolis, IN 46202, USA
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34
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Abstract
MicroRNAs (miRNAs) are integral to the gene regulatory network. A single miRNA is capable of controlling the expression of hundreds of protein coding genes and modulate a wide spectrum of biological functions, such as proliferation, differentiation, stress responses, DNA repair, cell adhesion, motility, inflammation, cell survival, senescence and apoptosis, all of which are fundamental to tumorigenesis. Overexpression, genetic amplification, and gain-of-function mutation of oncogenic miRNAs ("onco-miRs") as well as genetic deletion and loss-of-function mutation of tumor suppressor miRNAs ("suppressor-miRs") are linked to human cancer. In addition to the dysregulation of a specific onco-miR or suppressor-miRs, changes in global miRNA levels resulting from a defective miRNA biogenesis pathway play a role in tumorigenesis. The function of individual onco-miRs and suppressor-miRs and their target genes in cancer has been described in many different articles elsewhere. In this review, we primarily focus on the recent development regarding the dysregulation of the miRNA biogenesis pathway and its contribution to cancer.
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Affiliation(s)
- Akiko Hata
- a Cardiovascular Research Institute, University of California , San Francisco , CA , USA
| | - Risa Kashima
- a Cardiovascular Research Institute, University of California , San Francisco , CA , USA
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35
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Lee D, Goldberg AL. Muscle Wasting in Fasting Requires Activation of NF-κB and Inhibition of AKT/Mechanistic Target of Rapamycin (mTOR) by the Protein Acetylase, GCN5. J Biol Chem 2015; 290:30269-79. [PMID: 26515065 DOI: 10.1074/jbc.m115.685164] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2015] [Indexed: 12/25/2022] Open
Abstract
NF-κB is best known for its pro-inflammatory and anti-apoptotic actions, but in skeletal muscle, NF-κB activation is important for atrophy upon denervation or cancer. Here, we show that also upon fasting, NF-κB becomes activated in muscle and is critical for the subsequent atrophy. Following food deprivation, the expression and acetylation of the p65 of NF-κB on lysine 310 increase markedly in muscles. NF-κB inhibition in mouse muscles by overexpression of the IκBα superrepressor (IκBα-SR) or of p65 mutated at Lys-310 prevented atrophy. Knockdown of GCN5 with shRNA or a dominant-negative GCN5 or overexpression of SIRT1 decreased p65K310 acetylation and muscle wasting upon starvation. In addition to reducing atrogene expression, surprisingly inhibiting NF-κB with IκBα-SR or by GCN5 knockdown in these muscles also enhanced AKT and mechanistic target of rapamycin (mTOR) activities, which also contributed to the reduction in atrophy. These new roles of NF-κB and GCN5 in regulating muscle proteolysis and AKT/mTOR signaling suggest novel approaches to combat muscle wasting.
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Affiliation(s)
- Donghoon Lee
- From the Department of Cell Biology, Harvard Medical School, Boston, Masachusetts 02115
| | - Alfred L Goldberg
- From the Department of Cell Biology, Harvard Medical School, Boston, Masachusetts 02115
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36
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Libânio D, Dinis-Ribeiro M, Pimentel-Nunes P. Helicobacter pylori and microRNAs: Relation with innate immunity and progression of preneoplastic conditions. World J Clin Oncol 2015; 6:111-132. [PMID: 26468448 PMCID: PMC4600186 DOI: 10.5306/wjco.v6.i5.111] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/29/2015] [Revised: 06/22/2015] [Accepted: 08/04/2015] [Indexed: 02/06/2023] Open
Abstract
The accepted paradigm for intestinal-type gastric cancer pathogenesis is a multistep progression from chronic gastritis induced by Helicobacter pylori (H. pylori) to gastric atrophy, intestinal metaplasia, dysplasia and ultimately gastric cancer. The genetic and molecular mechanisms underlying disease progression are still not completely understood as only a fraction of colonized individuals ever develop neoplasia suggesting that bacterial, host and environmental factors are involved. MicroRNAs are noncoding RNAs that may influence H. pylori-related pathology through the regulation of the transcription and expression of various genes, playing an important role in inflammation, cell proliferation, apoptosis and differentiation. Indeed, H. pylori have been shown to modify microRNA expression in the gastric mucosa and microRNAs are involved in the immune host response to the bacteria and in the regulation of the inflammatory response. MicroRNAs have a key role in the regulation of inflammatory pathways and H. pylori may influence inflammation-mediated gastric carcinogenesis possibly through DNA methylation and epigenetic silencing of tumor suppressor microRNAs. Furthermore, microRNAs influenced by H. pylori also have been found to be involved in cell cycle regulation, apoptosis and epithelial-mesenchymal transition. Altogether, microRNAs seem to have an important role in the progression from gastritis to preneoplastic conditions and neoplastic lesions and since each microRNA can control the expression of hundreds to thousands of genes, knowledge of microRNAs target genes and their functions are of paramount importance. In this article we present a comprehensive review about the role of microRNAs in H. pylori gastric carcinogenesis, identifying the microRNAs downregulated and upregulated in the infection and clarifying their biological role in the link between immune host response, inflammation, DNA methylation and gastric carcinogenesis.
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37
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38
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Gurianova V, Stroy D, Kruzliak P, Kyrichenko V, Moibenko A, Dosenko V. Does proteasome regulate the level of microRNA-1 in cardiomyocytes? Application to anoxia-reoxygenation. Mol Cell Biochem 2015; 404:45-51. [PMID: 25724682 DOI: 10.1007/s11010-015-2365-7] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2014] [Accepted: 02/21/2015] [Indexed: 11/30/2022]
Abstract
Proteasome and microRNAs play a critical role in almost all processes in a living organism, including pathology of the heart; however, their interaction is still in question. In the present study, we have found that proteasome inhibitor provoked increase of mature but not immature microRNA-1 in cultured cardiomyocytes, and tested the hypothesis that mature microRNA-1 can be a substrate for endonuclease activity of proteasome. In our in vitro experiments, we have found that proteasome fraction II is able to degrade both mature and primary but not precursor microRNA-1. However, this in vitro effect was not abolished by chemical inhibitor of proteolytic activities of proteasome. These data let us summarize that proteasome has the complex effect on the level of microRNA-1.
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Affiliation(s)
- Veronika Gurianova
- Bogomoletz Institute of Physiology, National Academy of Science, Kiev, Ukraine
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39
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Zhou Y, Zhang N, Li BQ, Huang T, Cai YD, Kong XY. A method to distinguish between lysine acetylation and lysine ubiquitination with feature selection and analysis. J Biomol Struct Dyn 2015; 33:2479-90. [PMID: 25616595 DOI: 10.1080/07391102.2014.1001793] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Lysine acetylation and ubiquitination are two primary post-translational modifications (PTMs) in most eukaryotic proteins. Lysine residues are targets for both types of PTMs, resulting in different cellular roles. With the increasing availability of protein sequences and PTM data, it is challenging to distinguish the two types of PTMs on lysine residues. Experimental approaches are often laborious and time consuming. There is an urgent need for computational tools to distinguish between lysine acetylation and ubiquitination. In this study, we developed a novel method, called DAUFSA (distinguish between lysine acetylation and lysine ubiquitination with feature selection and analysis), to discriminate ubiquitinated and acetylated lysine residues. The method incorporated several types of features: PSSM (position-specific scoring matrix) conservation scores, amino acid factors, secondary structures, solvent accessibilities, and disorder scores. By using the mRMR (maximum relevance minimum redundancy) method and the IFS (incremental feature selection) method, an optimal feature set containing 290 features was selected from all incorporated features. A dagging-based classifier constructed by the optimal features achieved a classification accuracy of 69.53%, with an MCC of .3853. An optimal feature set analysis showed that the PSSM conservation score features and the amino acid factor features were the most important attributes, suggesting differences between acetylation and ubiquitination. Our study results also supported previous findings that different motifs were employed by acetylation and ubiquitination. The feature differences between the two modifications revealed in this study are worthy of experimental validation and further investigation.
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Affiliation(s)
- You Zhou
- a The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine , Shanghai 200031 , P.R. China
| | - Ning Zhang
- b Department of Biomedical Engineering, Tianjin Key Lab of BME Measurement , Tianjin University , Tianjin 300072 , P.R. China
| | - Bi-Qing Li
- c Key Laboratory of Systems Biology , Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai 200031 , P.R. China
| | - Tao Huang
- a The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine , Shanghai 200031 , P.R. China.,d Department of Genetics and Genomic Sciences , Icahn School of Medicine at Mount Sinai , New York , NY 10029 , USA
| | - Yu-Dong Cai
- e Institute of Systems Biology , Shanghai University , Shanghai 200444 , P.R. China
| | - Xiang-Yin Kong
- a The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine , Shanghai 200031 , P.R. China
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40
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Farooqi AA, Qureshi MZ, Coskunpinar E, Naqvi SKUH, Yaylim I, Ismail M. MiR-421, miR-155 and miR-650: emerging trends of regulation of cancer and apoptosis. Asian Pac J Cancer Prev 2014; 15:1909-12. [PMID: 24716910 DOI: 10.7314/apjcp.2014.15.5.1909] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022] Open
Abstract
It is becoming progressively more understandable that between transcription and translation there lies another versatile regulator that quantitatively controls the expression of mRNAs. Identification of miRNAs as key regulators of wide ranging signaling cascades and modulators of different cell-type and context dependent activities attracted basic and clinical scientists to study modes and mechanisms in details. In line with this approach overwhelmingly increasing in vivo and in vitro studies are deepening our understanding regarding miR-421, mir-155 and miR-650 mediated regulation of cellular activities. We also attempt to provide an overview of long non coding RNAs.
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Affiliation(s)
- Ammad Ahmad Farooqi
- Laboratory for Translational Oncology and Personalized Medicine, Rashid Latif Medical College, Lahore, Pakistan E-mail :
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Abstract
MicroRNAs (miRNAs) are small non-coding RNAs that function as guide molecules in RNA silencing. Targeting most protein-coding transcripts, miRNAs are involved in nearly all developmental and pathological processes in animals. The biogenesis of miRNAs is under tight temporal and spatial control, and their dysregulation is associated with many human diseases, particularly cancer. In animals, miRNAs are ∼22 nucleotides in length, and they are produced by two RNase III proteins--Drosha and Dicer. miRNA biogenesis is regulated at multiple levels, including at the level of miRNA transcription; its processing by Drosha and Dicer in the nucleus and cytoplasm, respectively; its modification by RNA editing, RNA methylation, uridylation and adenylation; Argonaute loading; and RNA decay. Non-canonical pathways for miRNA biogenesis, including those that are independent of Drosha or Dicer, are also emerging.
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Jee D, Lai EC. Alteration of miRNA activity via context-specific modifications of Argonaute proteins. Trends Cell Biol 2014; 24:546-53. [PMID: 24865524 DOI: 10.1016/j.tcb.2014.04.008] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2014] [Revised: 04/22/2014] [Accepted: 04/23/2014] [Indexed: 12/12/2022]
Abstract
miRNAs are enclosed within Argonaute (Ago) proteins, the downstream effectors of small RNA-mediated gene silencing. Because miRNAs mediate extensive networks of post-transcriptional control, cells have evolved multiple strategies to control their activity with precision. A growing theme of recent years is how post-translational modifications of Ago proteins, such as prolyl hydroxylation, phosphorylation, ubiquitination, and poly-ADP-ribosylation, alter miRNA activity at global or specific levels. In this review, we discuss recent advances in Ago modifications in mammalian cells and emphasize how such alterations modulate small RNA function to coordinate appropriate downstream cellular responses. These findings provide a framework to understand how Ago protein modifications are linked to reorganization of post-transcriptional regulatory networks, enabling dynamic responses to diverse external stimuli and changing environmental conditions.
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Affiliation(s)
- David Jee
- Department of Developmental Biology, Sloan-Kettering Institute, 1275 York Ave, Box 252, New York, NY 10065, USA; Program in Biochemistry, Cell and Molecular Biology, Weill Cornell Graduate School of Medical Sciences, New York, NY 10065, USA
| | - Eric C Lai
- Department of Developmental Biology, Sloan-Kettering Institute, 1275 York Ave, Box 252, New York, NY 10065, USA.
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Phosphorylation of DGCR8 increases its intracellular stability and induces a progrowth miRNA profile. Cell Rep 2013; 5:1070-81. [PMID: 24239349 PMCID: PMC3892995 DOI: 10.1016/j.celrep.2013.10.017] [Citation(s) in RCA: 74] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2012] [Revised: 07/03/2013] [Accepted: 10/09/2013] [Indexed: 02/07/2023] Open
Abstract
During miRNA biogenesis, the microprocessor complex (MC), which is composed minimally of Drosha, an RNase III enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary miRNA (pri-miRNA) in order to release the pre-miRNA stem-loop structure. Using phosphoproteomics, we mapped 23 phosphorylation sites on full-length human DGCR8 expressed in insect or mammalian cells. DGCR8 can be phosphorylated by mitogenic ERK/MAPK, indicating that DGCR8 phosphorylation may respond to and integrate extracellular cues. The expression of phosphomimetic DGCR8 or inhibition of phosphatases increased the cellular levels of DGCR8 and Drosha proteins. Increased levels of phosphomimetic DGCR8 were not due to higher mRNA levels, altered DGCR8 localization, or DGCR8’s ability to self-associate, but rather to an increase in protein stability. MCs incorporating phosphomutant or phosphomimetic DGCR8 were not altered in specific processing activity. However, HeLa cells expressing phosphomimetic DGCR8 exhibited a progrowth miRNA expression profile and increased proliferation and scratch closure rates relative to cells expressing phosphomutant DGCR8.
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