|
1
|
Li Z, Liao Y, Tang C, Xu L, Peng B, Xu X. RBM14 promotes DNA end resection during homologous recombination repair. Acta Biochim Biophys Sin (Shanghai) 2023; 55:1864-1873. [PMID: 37559455 PMCID: PMC10753362 DOI: 10.3724/abbs.2023104] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2023] [Accepted: 05/25/2023] [Indexed: 08/11/2023] Open
Abstract
DNA double-strand break (DSB) repair by homologous recombination (HR) is crucial for the maintenance of genome stability and integrity. In this study, we aim to identify novel RNA binding proteins (RBPs) involved in HR repair because little is known about RBP function in HR. For this purpose, we carry out pulldown assays using a synthetic ssDNA/dsDNA structure coated with replication protein A (RPA) to mimic resected DNA, a crucial intermediate in HR-mediated DSB repair. Using this approach, we identify RNA-binding motif protein 14 (RBM14) as a potential binding partner. We further show that RBM14 interacts with an essential HR repair factor, CtIP. RBM14 is crucial for CtIP recruitment to DSB sites and for subsequent RPA coating and RAD51 replacement, facilitating efficient HR repair. Moreover, inhibition of RBM14 expression sensitizes cancer cells to X-ray irradiation. Together, our results demonstrate that RBM14 promotes DNA end resection to ensure HR repair and may serve as a potential target for cancer therapy.
Collapse
Affiliation(s)
- Zheng Li
- College of Life SciencesCapital Normal UniversityBeijing100048China
- Guangdong Key Laboratory for Genome Stability & Disease Prevention and Carson International Cancer CenterMarshall Laboratory of Biomedical EngineeringShenzhen University Medical SchoolShenzhen UniversityShenzhen518060China
| | - Yanting Liao
- Guangdong Key Laboratory for Genome Stability & Disease Prevention and Carson International Cancer CenterMarshall Laboratory of Biomedical EngineeringShenzhen University Medical SchoolShenzhen UniversityShenzhen518060China
| | - Chen Tang
- State Key Laboratory of Agro-biotechnology and MOA Key Laboratory of Soil MicrobiologyCollege of Biological SciencesChina Agricultural UniversityBeijing100091China
- Shenzhen University General Hospital-Dehua Hospital Joint Research Center on Precision Medicine (sgh-dhhCPM)Dehua HospitalDehua362500China
| | - Linli Xu
- College of Life SciencesCapital Normal UniversityBeijing100048China
| | - Bin Peng
- Guangdong Key Laboratory for Genome Stability & Disease Prevention and Carson International Cancer CenterMarshall Laboratory of Biomedical EngineeringShenzhen University Medical SchoolShenzhen UniversityShenzhen518060China
| | - Xingzhi Xu
- Guangdong Key Laboratory for Genome Stability & Disease Prevention and Carson International Cancer CenterMarshall Laboratory of Biomedical EngineeringShenzhen University Medical SchoolShenzhen UniversityShenzhen518060China
| |
Collapse
|
|
2
|
Wang X, Ma T, Wei C, Liu J, Yu T, Zou Y, Liu S, Yang Z, Xi J. Toxic effects of exogenous retinoic acid on the neurodevelopment of zebrafish (Danio rerio) embryos. Neurotoxicol Teratol 2023; 100:107291. [PMID: 37689270 DOI: 10.1016/j.ntt.2023.107291] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2023] [Revised: 08/30/2023] [Accepted: 08/30/2023] [Indexed: 09/11/2023]
Abstract
Endogenous retinoic acid (RA) is essential for embryonic development and maintaining adult physiological processes. Human-caused RA residues in the environment threaten the survival of organisms in the environment. We employed zebrafish as a model to explore the developmental impacts of excess RA. We used exogenous RA to raise the amount of RA signal in the embryos and looked at the effects of excess RA on embryonic morphological development. Upregulation of the RA signal significantly reduced embryo hatching and increased embryo malformation. To further understand the neurotoxic impact of RA signaling on early neurodevelopment, we measured the expression of neurodevelopmental marker genes and cell death and proliferation markers in zebrafish embryos. Exogenous RA disrupted stem cell (SC) and neuron marker gene expression and exacerbated apoptosis in the embryos. Furthermore, we looked into the links between the transcriptional coactivator RBM14 and RA signaling to better understand the mechanism of RA neurotoxicity. There was a negative interaction between RA signaling and the transcription coactivator RBM14, and the morpholino-induced RBM14 down-regulation can partially block the effects of RAR antagonist BMS493-induced RA signaling inhibition on embryonic malformation and cell apoptosis. In conclusion, exogenous RA causes neurodevelopmental toxicity, and RBM14 may be involved in this neurotoxic process.
Collapse
Affiliation(s)
- Xiaoxuan Wang
- Institute of Pharmaceutical Innovation, Hubei Province Key Laboratory of Occupational Hazard Identification and Control, School of Medicine, Wuhan University of Science and Technology, Wuhan, China
| | - Ting Ma
- Institute of Pharmaceutical Innovation, Hubei Province Key Laboratory of Occupational Hazard Identification and Control, School of Medicine, Wuhan University of Science and Technology, Wuhan, China
| | - Cizhao Wei
- Demonstration Center for Experimental Basic Medicine Education, Wuhan University Taikang Medical School (School of Basic Medical Sciences), Wuhan, Hubei, China
| | - Juan Liu
- Institute of Pharmaceutical Innovation, Hubei Province Key Laboratory of Occupational Hazard Identification and Control, School of Medicine, Wuhan University of Science and Technology, Wuhan, China
| | - Ting Yu
- Institute of Pharmaceutical Innovation, Hubei Province Key Laboratory of Occupational Hazard Identification and Control, School of Medicine, Wuhan University of Science and Technology, Wuhan, China
| | - Yu Zou
- Institute of Pharmaceutical Innovation, Hubei Province Key Laboratory of Occupational Hazard Identification and Control, School of Medicine, Wuhan University of Science and Technology, Wuhan, China
| | - Song Liu
- Institute of Pharmaceutical Innovation, Hubei Province Key Laboratory of Occupational Hazard Identification and Control, School of Medicine, Wuhan University of Science and Technology, Wuhan, China
| | - Zheqiong Yang
- Demonstration Center for Experimental Basic Medicine Education, Wuhan University Taikang Medical School (School of Basic Medical Sciences), Wuhan, Hubei, China.
| | - Jinlei Xi
- Institute of Pharmaceutical Innovation, Hubei Province Key Laboratory of Occupational Hazard Identification and Control, School of Medicine, Wuhan University of Science and Technology, Wuhan, China.
| |
Collapse
|
|
3
|
Hu J, Yang L, Peng X, Mao M, Liu X, Song J, Li H, Chen F. METTL3 promotes m6A hypermethylation of RBM14 via YTHDF1 leading to the progression of hepatocellular carcinoma. Hum Cell 2022; 35:1838-1855. [PMID: 36087219 DOI: 10.1007/s13577-022-00769-3] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2022] [Accepted: 08/09/2022] [Indexed: 11/27/2022]
Abstract
Liver is a well-known immunological organ with unique microenvironment. In normal conditions, the rich immune-infiltrating cells cooperate with non-parenchymal cells, such as Kupffer cells (KCs). The presence of liver immunosuppressive microenvironment underlines the importance to dissect this interaction to understand how this cross-talk promotes tumor growth in hepatocellular carcinoma (HCC). Therefore, the aim of the study here was to probe the role of methyltransferase-like 3 (METTL3) in the HCC progression and its effect on the polarization of KCs. KCs showed M2 polarization, and METTL3 was overexpressed in our collected HCC tissues relative to adjacent tissues. METTL3 depletion inhibited the M2 polarization of KCs, thereby reverting the malignant phenotype of HCC cells in vitro and growth and metastasis in vivo. Mechanistically, YTH domain-containing family protein 1 (YTHDF1) bound to RNA-binding protein 14 (RBM14), whereas METTL3 knockdown in KCs cells suppressed RBM14 expression by decreasing N-methyladenosine (m6A) methylation. Overexpression of RBM14 mitigated the anti-tumor effects of sh-METTL3 in vitro and in vivo. It is suggested that the mechanism of sh-METTL3 suppressing the polarization of KCs and the progression of HCC is to regulate the RBM14 expression via YTHDF1-dependent m6A modification.
Collapse
Affiliation(s)
- Jingyao Hu
- The Seventh Department of General Surgery, The Fourth Affiliated Hospital of China Medical University, No. 4, Donggu Road, Chonggu District, Shenyang, 110000, Liaoning, People's Republic of China
| | - Liang Yang
- The Third Department of General Surgery, The Fourth Affiliated Hospital of China Medical University, Shenyang, 110000, Liaoning, People's Republic of China
| | - Xueqiang Peng
- The Third Department of General Surgery, The Fourth Affiliated Hospital of China Medical University, Shenyang, 110000, Liaoning, People's Republic of China
| | - Minghuan Mao
- Department of Urology Surgery, The Fourth Affiliated Hospital of China Medical University, Shenyang, 110000, Liaoning, People's Republic of China
| | - Xiaodan Liu
- The Fifth Department of General Surgery, The Fourth Affiliated Hospital of China Medical University, Shenyang, 110000, Liaoning, People's Republic of China
| | - Jianbo Song
- Interventional Department, The Fourth Affiliated Hospital of China Medical University, Shenyang, 110000, Liaoning, People's Republic of China
| | - Hangyu Li
- The Third Department of General Surgery, The Fourth Affiliated Hospital of China Medical University, Shenyang, 110000, Liaoning, People's Republic of China.
| | - Fu Chen
- The Seventh Department of General Surgery, The Fourth Affiliated Hospital of China Medical University, No. 4, Donggu Road, Chonggu District, Shenyang, 110000, Liaoning, People's Republic of China.
| |
Collapse
|
|
4
|
Ebrahimie E, Rahimirad S, Tahsili M, Mohammadi-Dehcheshmeh M. Alternative RNA splicing in stem cells and cancer stem cells: Importance of transcript-based expression analysis. World J Stem Cells 2021; 13:1394-1416. [PMID: 34786151 PMCID: PMC8567453 DOI: 10.4252/wjsc.v13.i10.1394] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/18/2021] [Revised: 06/21/2021] [Accepted: 09/14/2021] [Indexed: 02/06/2023] Open
Abstract
Alternative ribonucleic acid (RNA) splicing can lead to the assembly of different protein isoforms with distinctive functions. The outcome of alternative splicing (AS) can result in a complete loss of function or the acquisition of new functions. There is a gap in knowledge of abnormal RNA splice variants promoting cancer stem cells (CSCs), and their prospective contribution in cancer progression. AS directly regulates the self-renewal features of stem cells (SCs) and stem-like cancer cells. Notably, octamer-binding transcription factor 4A spliced variant of octamer-binding transcription factor 4 contributes to maintaining stemness properties in both SCs and CSCs. The epithelial to mesenchymal transition pathway regulates the AS events in CSCs to maintain stemness. The alternative spliced variants of CSCs markers, including cluster of differentiation 44, aldehyde dehydrogenase, and doublecortin-like kinase, α6β1 integrin, have pivotal roles in increasing self-renewal properties and maintaining the pluripotency of CSCs. Various splicing analysis tools are considered in this study. LeafCutter software can be considered as the best tool for differential splicing analysis and identification of the type of splicing events. Additionally, LeafCutter can be used for efficient mapping splicing quantitative trait loci. Altogether, the accumulating evidence re-enforces the fact that gene and protein expression need to be investigated in parallel with alternative splice variants.
Collapse
Affiliation(s)
- Esmaeil Ebrahimie
- School of Animal and Veterinary Sciences, The University of Adelaide, Adelaide 5005, South Australia, Australia
- La Trobe Genomics Research Platform, School of Life Sciences, College of Science, Health and Engineering, La Trobe University, Melbourne 3086, Australia
- School of Biosciences, The University of Melbourne, Melbourne 3010, Australia,
| | - Samira Rahimirad
- Department of Medical Genetics, National Institute of Genetic Engineering and Biotechnology, Tehran 1497716316, Iran
- Division of Urology, Department of Surgery, McGill University and the Research Institute of the McGill University Health Centre, Montreal H4A 3J1, Quebec, Canada
| | | | | |
Collapse
|
|
5
|
Xu (徐兴丽) X, Xu (徐兴华) X, Mao (毛洋) Y, Lu (卢琳) L, Ma (马静) J, Zheng (郑腾飞) T, Zhang (张杰) J, Zhang (章萌) M, Meng (孟霖霖) L, Ma (马连越) L, Cheng (程晶) J, Chen (陈文强) W, Jiang (姜虹) H, Zhang (张运) Y, Zhang (张澄) C. Knockout of the NONO Gene Inhibits Neointima Formation in a Mouse Model of Vascular Injury. Arterioscler Thromb Vasc Biol 2021; 41:1428-1445. [PMID: 33626912 DOI: 10.1161/atvbaha.119.313581] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
[Figure: see text].
Collapse
Affiliation(s)
- Xingli Xu (徐兴丽)
- Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China (Xingli Xu, Xinghua Xu, Y.M., L.L., J.M., T.Z., J.Z., M.Z., L. Meng, L. Ma, J.C., W.C., H.J., Y.Z., C.Z.).,Ultrasound in Cardiac Electrophysiology and Biomechanics Key Laboratory of Sichuan Province, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu (Xingli Xu).,Chinese Academy of Sciences Sichuan Translational Medicine Research Hospital, Chengdu, China (Xingli Xu)
| | - Xinghua Xu (徐兴华)
- Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China (Xingli Xu, Xinghua Xu, Y.M., L.L., J.M., T.Z., J.Z., M.Z., L. Meng, L. Ma, J.C., W.C., H.J., Y.Z., C.Z.).,Department of Histology and Embryology, Shandong First Medical University and Shandong Academy of Medical Science, Taian, China (Xinghua Xu)
| | - Yang Mao (毛洋)
- Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China (Xingli Xu, Xinghua Xu, Y.M., L.L., J.M., T.Z., J.Z., M.Z., L. Meng, L. Ma, J.C., W.C., H.J., Y.Z., C.Z.)
| | - Lin Lu (卢琳)
- Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China (Xingli Xu, Xinghua Xu, Y.M., L.L., J.M., T.Z., J.Z., M.Z., L. Meng, L. Ma, J.C., W.C., H.J., Y.Z., C.Z.)
| | - Jing Ma (马静)
- Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China (Xingli Xu, Xinghua Xu, Y.M., L.L., J.M., T.Z., J.Z., M.Z., L. Meng, L. Ma, J.C., W.C., H.J., Y.Z., C.Z.)
| | - Tengfei Zheng (郑腾飞)
- Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China (Xingli Xu, Xinghua Xu, Y.M., L.L., J.M., T.Z., J.Z., M.Z., L. Meng, L. Ma, J.C., W.C., H.J., Y.Z., C.Z.)
| | - Jie Zhang (张杰)
- Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China (Xingli Xu, Xinghua Xu, Y.M., L.L., J.M., T.Z., J.Z., M.Z., L. Meng, L. Ma, J.C., W.C., H.J., Y.Z., C.Z.)
| | - Meng Zhang (章萌)
- Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China (Xingli Xu, Xinghua Xu, Y.M., L.L., J.M., T.Z., J.Z., M.Z., L. Meng, L. Ma, J.C., W.C., H.J., Y.Z., C.Z.)
| | - Linlin Meng (孟霖霖)
- Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China (Xingli Xu, Xinghua Xu, Y.M., L.L., J.M., T.Z., J.Z., M.Z., L. Meng, L. Ma, J.C., W.C., H.J., Y.Z., C.Z.)
| | - Lianyue Ma (马连越)
- Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China (Xingli Xu, Xinghua Xu, Y.M., L.L., J.M., T.Z., J.Z., M.Z., L. Meng, L. Ma, J.C., W.C., H.J., Y.Z., C.Z.)
| | - Jing Cheng (程晶)
- Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China (Xingli Xu, Xinghua Xu, Y.M., L.L., J.M., T.Z., J.Z., M.Z., L. Meng, L. Ma, J.C., W.C., H.J., Y.Z., C.Z.)
| | - Wenqiang Chen (陈文强)
- Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China (Xingli Xu, Xinghua Xu, Y.M., L.L., J.M., T.Z., J.Z., M.Z., L. Meng, L. Ma, J.C., W.C., H.J., Y.Z., C.Z.)
| | - Hong Jiang (姜虹)
- Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China (Xingli Xu, Xinghua Xu, Y.M., L.L., J.M., T.Z., J.Z., M.Z., L. Meng, L. Ma, J.C., W.C., H.J., Y.Z., C.Z.)
| | - Yun Zhang (张运)
- Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China (Xingli Xu, Xinghua Xu, Y.M., L.L., J.M., T.Z., J.Z., M.Z., L. Meng, L. Ma, J.C., W.C., H.J., Y.Z., C.Z.)
| | - Cheng Zhang (张澄)
- Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education, Chinese National Health Commission and Chinese Academy of Medical Sciences, The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital of Shandong University, Jinan, China (Xingli Xu, Xinghua Xu, Y.M., L.L., J.M., T.Z., J.Z., M.Z., L. Meng, L. Ma, J.C., W.C., H.J., Y.Z., C.Z.)
| |
Collapse
|
|
6
|
Chan HC, Lau YT, Ding Q, Li CK, Wong CM, Shaw PC, Waye MMY, Tsang SY. PinX1t, a Novel PinX1 Transcript Variant, Positively Regulates Cardiogenesis of Embryonic Stem Cells. J Am Heart Assoc 2020; 9:e010240. [PMID: 32157956 PMCID: PMC7335523 DOI: 10.1161/jaha.118.010240] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Background Pin2/TRF1‐interacting protein, PinX1, was previously identified as a tumor suppressor. Here, we discovered a novel transcript variant of mPinX1 (mouse PinX1), mPinX1t (mouse PinX1t), in embryonic stem cells (ESCs). The aims of this investigation were (1) to detect the presence of mPinX1 and mPinX1t in ESCs and their differentiation derivatives; (2) to investigate the role of mPinX1 and mPinX1t on regulating the characteristics of undifferentiated ESCs and the cardiac differentiation of ESCs; (3) to elucidate the molecular mechanisms of how mPinX1 and mPinX1t regulate the cardiac differentiation of ESCs. Methods and Results By 5′ rapid amplification of cDNA ends, 3′ rapid amplification of cDNA ends, and polysome fractionation followed by reverse transcription–polymerase chain reaction, mPinX1t transcript was confirmed to be an intact mRNA that is actively translated. Western blot confirmed the existence of mPinX1t protein. Overexpression or knockdown of mPinX1 (both decreased mPinX1t expression) both decreased while overexpression of mPinX1t increased the cardiac differentiation of ESCs. Although both mPinX1 and mPinX1t proteins were found to bind to cardiac transcription factor mRNAs, only mPinX1t protein but not mPinX1 protein was found to bind to nucleoporin 133 protein, a nuclear pore complex component. In addition, mPinX1t‐containing cells were found to have a higher cytosol‐to‐nucleus ratio of cardiac transcription factor mRNAs when compared with that in the control cells. Our data suggested that mPinX1t may positively regulate cardiac differentiation by enhancing export of cardiac transcription factor mRNAs through interacting with nucleoporin 133. Conclusions We discovered a novel transcript variant of mPinX1, the mPinX1t, which positively regulates the cardiac differentiation of ESCs.
Collapse
Affiliation(s)
- Hing Chung Chan
- School of Life Sciences The Chinese University of Hong Kong Hong Kong SAR
| | - Yuen Ting Lau
- School of Life Sciences The Chinese University of Hong Kong Hong Kong SAR
| | - Qianqian Ding
- School of Life Sciences The Chinese University of Hong Kong Hong Kong SAR
| | - Chun Kit Li
- School of Life Sciences The Chinese University of Hong Kong Hong Kong SAR
| | - Chi Ming Wong
- Department of Health Technology and Informatics The Hong Kong Polytechnic University Hong Kong SAR
| | - Pang Chui Shaw
- School of Life Sciences The Chinese University of Hong Kong Hong Kong SAR
| | - Mary Miu Yee Waye
- The Nethersole School of Nursing The Chinese University of Hong Kong Hong Kong SAR.,The Croucher Laboratory for Human Genomics The Chinese University of Hong Kong Hong Kong SAR
| | - Suk Ying Tsang
- School of Life Sciences The Chinese University of Hong Kong Hong Kong SAR.,State Key Laboratory of Agrobiotechnology The Chinese University of Hong Kong Hong Kong SAR.,Key Laboratory for Regenerative Medicine Ministry of Education The Chinese University of Hong Kong Hong Kong SAR.,Centre for Novel Biomaterials The Chinese University of Hong Kong Hong Kong SAR
| |
Collapse
|
|
7
|
Xiao Y, Chen J, Wan Y, Gao Q, Jing N, Zheng Y, Zhu X. Regulation of zebrafish dorsoventral patterning by phase separation of RNA-binding protein Rbm14. Cell Discov 2019; 5:37. [PMID: 31636951 PMCID: PMC6796953 DOI: 10.1038/s41421-019-0106-x] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2019] [Revised: 06/13/2019] [Accepted: 06/14/2019] [Indexed: 02/08/2023] Open
Abstract
RNA-binding proteins with intrinsically disordered regions (IDRs) such as Rbm14 can phase separate in vitro. To what extent the phase separation contributes to their physiological functions is however unclear. Here we show that zebrafish Rbm14 regulates embryonic dorsoventral patterning through phase separation. Zebrafish rbm14 morphants displayed dorsalized phenotypes associated with attenuated BMP signaling. Consistently, depletion of mammalian Rbm14 downregulated BMP regulators and effectors Nanog, Smad4/5, and Id1/2, whereas overexpression of the BMP-related proteins in the morphants significantly restored the developmental defects. Importantly, the IDR of zebrafish Rbm14 demixed into liquid droplets in vitro despite poor sequence conservation with its mammalian counterpart. While its phase separation mutants or IDR failed to rescue the morphants, its chimeric proteins containing an IDR from divergent phase separation proteins were effective. Rbm14 complexed with proteins involved in RNA metabolism and phase separated into cellular ribonucleoprotein compartments. Consistently, RNA deep sequencing analysis on the morphant embryos revealed increased alternative splicing events as well as large-scale transcriptomic downregulations. Our results suggest that Rbm14 functions in ribonucleoprotein compartments through phase separation to modulate multiple aspects of RNA metabolism. Furthermore, IDRs conserve in phase separation ability but not primary sequence and can be functionally interchangeable.
Collapse
Affiliation(s)
- Yue Xiao
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 320 Yueyang Road, 200031 Shanghai, China
| | - Jiehui Chen
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 320 Yueyang Road, 200031 Shanghai, China
| | - Yihan Wan
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 320 Yueyang Road, 200031 Shanghai, China
- Department of Embryology, Carnegie Institution for Science, 3520 San Martin Dr., Baltimore, MD 21218 USA
| | - Qi Gao
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 320 Yueyang Road, 200031 Shanghai, China
| | - Naihe Jing
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 320 Yueyang Road, 200031 Shanghai, China
| | - Yixian Zheng
- Department of Embryology, Carnegie Institution for Science, 3520 San Martin Dr., Baltimore, MD 21218 USA
| | - Xueliang Zhu
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, 320 Yueyang Road, 200031 Shanghai, China
| |
Collapse
|
|
8
|
Chen G, Zhang D, Zhang L, Feng G, Zhang B, Wu Y, Li W, Zhang Y, Hu B. RBM14 is indispensable for pluripotency maintenance and mesoderm development of mouse embryonic stem cells. Biochem Biophys Res Commun 2018; 501:259-265. [PMID: 29729270 DOI: 10.1016/j.bbrc.2018.04.231] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2018] [Accepted: 04/30/2018] [Indexed: 12/31/2022]
Abstract
The pluripotency of embryonic stem cells (ESCs) is maintained by core pluripotency transcription factors, cofactors and several signaling pathways. RBM14 is a component of the para-speckle complex, which has been implicated in multiple important biological processes. The role of RBM14 in ESCs and lineage differentiation remains to be elucidated. In the present study, we provided evidence that RBM14 plays important roles in maintaining pluripotency and in the early differentiation of ESCs. RBM14 was demonstrated to be expressed in mouse embryonic stem cells (mESCs) and localized in the nucleus. RBM14 expression was depleted in mESCs using clustered regularly interspaced short palindromic repeats (CRISPR) technology. Our results also showed that RBM14 depletion altered the gene expression profiles of mESCs. In particular, pluripotency-associated genes and genes involved in the Wnt and TGF-β signaling pathways were downregulated in RBM14 knockout mESCs. Furthermore, RBM14 was found to be essential for mesoderm development in vitro and in vivo. The specific effects of RBM14 depletion were verified by conducting a rescue experiment. Our findings demonstrated that RBM14 not only plays an important role in maintaining the pluripotency of mESCs but is also indispensable for mesoderm development.
Collapse
Affiliation(s)
- Guilai Chen
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100101, China
| | - Da Zhang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
| | - Linlin Zhang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100101, China
| | - Guihai Feng
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100101, China
| | - Boya Zhang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China
| | - Yihui Wu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100101, China
| | - Wei Li
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100101, China
| | - Ying Zhang
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100101, China.
| | - Baoyang Hu
- State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China; University of Chinese Academy of Sciences, Beijing, 100101, China.
| |
Collapse
|
|
9
|
Yuan M, Eberhart CG, Kai M. RNA binding protein RBM14 promotes radio-resistance in glioblastoma by regulating DNA repair and cell differentiation. Oncotarget 2015; 5:2820-6. [PMID: 24811242 PMCID: PMC4058047 DOI: 10.18632/oncotarget.1924] [Citation(s) in RCA: 40] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023] Open
Abstract
Glioblastoma multiforme (GBM) is the most aggressive and lethal type of brain tumor. Standard treatment for GBM patients is surgery followed by radiotherapy and/or chemotherapy, but tumors always recur. Traditional therapies seem to fail because they eliminate only the bulk of the tumors and spare a population of stem-like cells termed tumor-initiating cells. The stem-like state and preferential activation of DNA damage response in the GBM tumor-initiating cells contribute to their radio-resistance and recurrence. The molecular mechanisms underlying this efficient activation of damage response and maintenance of stem-like state remain elusive. Here we show that RBM14 controls DNA repair pathways and also prevents cell differentiation in GBM spheres, causing radio-resistance. Knockdown of RBM14 affects GBM sphere maintenance and sensitizes radio-resistant GBM cells at the cellular level. We demonstrate that RBM14 knockdown blocks GBM regrowth after irradiation in vivo. In addition, RBM14 stimulates DNA repair by controlling the DNA-PK-dependent non-homologous end-joining (NHEJ) pathway. These results reveal unexpected functions of the RNA-binding protein RBM14 in control of DNA repair and maintenance of tumor-initiating cells. Targeting the RBM14-dependent pathway may prevent recurrence of tumors and eradicate the deadly disease completely.
Collapse
Affiliation(s)
- Ming Yuan
- Department of Radiation Oncology, Johns Hopkins University School of Medicine, Baltimore MD, USA
| | | | | |
Collapse
|
|
10
|
Trosko JE. Human adult stem cells as the target cells for the initiation of carcinogenesis and for the generation of "cancer stem cells". Int J Stem Cells 2014; 1:8-26. [PMID: 24855504 DOI: 10.15283/ijsc.2008.1.1.8] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/01/2008] [Indexed: 01/12/2023] Open
Abstract
The inference to stem cells has been found in ancient myths and the concept of stem cells has existed in the fields of plant biology, developmental biology and embryology for decades. In the field of cancer research, the stem cell theory was one of the earliest hypotheses on the origin of a cancer from a single cell. However, an opposing hypothesis had it that an adult differentiated somatic cell could "de-differentiate" to become a cancer cell. Only within the last decade, via the "cloning" of Dolly, the sheep, did the field of stem cell biology really trigger an exciting revolution in biological research. The isolation of human embryonic stem cells has created a true revolution in the life sciences that has led to the hope that these human stem cells could lead to (a) basic science understanding of gene regulation during differentiation and development; (b) stem cell therapy; (c) gene therapy via stem cells; (d) the use of stem cells for drug discovery; (e) screening for toxic effects of chemicals; and (f) understand the aging and diseases of aging processes.
Collapse
Affiliation(s)
- James E Trosko
- Department of Pediatrics/Human Development, College of Human Medicine, Michigan State University, East Lansing, Michigan 48824, USA
| |
Collapse
|
|
11
|
Ferrarese R, Harsh GR, Yadav AK, Bug E, Maticzka D, Reichardt W, Dombrowski SM, Miller TE, Masilamani AP, Dai F, Kim H, Hadler M, Scholtens DM, Yu ILY, Beck J, Srinivasasainagendra V, Costa F, Baxan N, Pfeifer D, von Elverfeldt D, Backofen R, Weyerbrock A, Duarte CW, He X, Prinz M, Chandler JP, Vogel H, Chakravarti A, Rich JN, Carro MS, Bredel M. Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression. J Clin Invest 2014; 124:2861-76. [PMID: 24865424 DOI: 10.1172/jci68836] [Citation(s) in RCA: 70] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2013] [Accepted: 04/03/2014] [Indexed: 01/08/2023] Open
Abstract
Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones.
Collapse
|
|
12
|
Peng M, Yang Z, Zhang H, Jaafar L, Wang G, Liu M, Flores-Rozas H, Xu J, Mivechi NF, Ko L. GT198 Splice Variants Display Dominant-Negative Activities and Are Induced by Inactivating Mutations. Genes Cancer 2013; 4:26-38. [PMID: 23946869 DOI: 10.1177/1947601913486345] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2012] [Accepted: 03/16/2013] [Indexed: 11/16/2022] Open
Abstract
Alternative pre-mRNA splicing yields functionally distinct splice variants in regulating normal cell differentiation as well as cancer development. The putative tumor suppressor gene GT198 (PSMC3IP), encoding a protein also known as TBPIP and Hop2, has been shown to regulate steroid hormone receptor-mediated transcription and to stimulate homologous recombination in DNA repair. Here, we have identified 6 distinct GT198 splice variant transcripts generated by alternative promoter usage or alternative splicing. Various splice variant transcripts preserve a common open reading frame, which encodes the DNA binding domain of GT198. The splice variants act as dominant negatives to counteract wild-type GT198 activity in transcription and to abolish Rad51 foci formation during radiation-induced DNA damage. In fallopian tube cancer, we have identified 44 point mutations in GT198 clustered in 2 mutation hotspot sequences. The mutation hotspots coincide with the regulatory sequences responsible for alternative splicing, strongly supporting that imbalanced alternative splicing is a selected consequence in cancer. In addition, splice variant-associated cytoplasmic expression is found in tumors carrying germline or somatic GT198 mutations. An altered alternative splicing pattern with increased variants is also present in lymphoblastoid cells derived from familial breast cancer patients carrying GT198 germline mutations. Furthermore, GT198 and its variant are reciprocally expressed during mouse stem cell differentiation. The constitutive expression of the GT198 variant but not the wild type induces tumor growth in nude mice. Our results collectively suggest that mutations in the GT198 gene deregulate alternative splicing. Defective alternative splicing promotes antagonizing variants and in turn induces a loss of the wild type in tumorigenesis. The study highlights the role of alternative splicing in tumor suppressor gene inactivation.
Collapse
Affiliation(s)
- Min Peng
- Cancer Center, Institute of Molecular Medicine and Genetics, Medical College of Georgia, Georgia Regents University, Augusta, GA, USA
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
13
|
The coactivator activator CoAA regulates PEA3 group member transcriptional activity. Biochem J 2011; 439:469-77. [PMID: 21736557 DOI: 10.1042/bj20110728] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The PEA3 (polyoma enhancer activator 3) group members [ERM (ETS-related molecule), ER81 (ETS-related 81) and PEA3] of the Ets transcription factor family are involved in migration and dissemination processes during organogenesis and cancer development. In the present study, we report that the hnRNP (heterogeneous nuclear ribonucleoprotein)-like protein CoAA (Coactivator activator) interacts with the PEA3 group members and modulates their transcriptional activity. We also demonstrate that the CoAA YQ domain, containing tyrosine/glutamine-rich hexapeptide repeats, is necessary for the interaction, whereas the two N-terminal RRMs (RNA recognition motifs) of CoAA are required to enhance transcriptional activity. Finally, we show that CoAA is involved in the migration-enhancing action of PEA3 on MCF7 human cancer cells, suggesting that CoAA might be an important regulator of PEA3 group member activity during metastasis.
Collapse
|
|
14
|
Ray P, Kar A, Fushimi K, Havlioglu N, Chen X, Wu JY. PSF suppresses tau exon 10 inclusion by interacting with a stem-loop structure downstream of exon 10. J Mol Neurosci 2011; 45:453-66. [PMID: 21881826 DOI: 10.1007/s12031-011-9634-z] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2011] [Accepted: 08/17/2011] [Indexed: 01/24/2023]
Abstract
Microtubule binding protein Tau has been implicated in a wide range of neurodegenerative disorders collectively classified as tauopathies. Exon 10 of the human tau gene, which codes for a microtubule binding repeat region, is alternatively spliced to form Tau protein isoforms containing either four or three microtubule binding repeats, Tau4R and Tau3R, respectively. The levels of different Tau splicing isoforms are fine-tuned by alternative splicing with the ratio of Tau4R/Tau3R maintained approximately at one in adult neurons. Mutations that disrupt tau exon 10 splicing regulation cause an imbalance of different tau splicing isoforms and have been associated with tauopathy. To search for factors interacting with tau pre-messenger RNA (pre-mRNA) and regulating tau exon 10 alternative splicing, we performed a yeast RNA-protein interaction screen and identified polypyrimidine tract binding protein associated splicing factor (PSF) as a candidate tau exon 10 splicing regulator. UV crosslinking experiments show that PSF binds to the stem-loop structure at the 5' splice site downstream of tau exon 10. This PSF-interacting RNA element is distinct from known PSF binding sites previously identified in other genes. Overexpression of PSF promotes tau exon 10 exclusion, whereas down-regulation of the endogenous PSF facilitates exon 10 inclusion. Immunostaining shows that PSF is expressed in the human brain regions affected by tauopathy. Our data reveal a new player in tau exon 10 alternative splicing regulation and uncover a previously unknown mechanism of PSF in regulating tau pre-mRNA splicing.
Collapse
Affiliation(s)
- Payal Ray
- Department of Neurology, Lurie Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
| | | | | | | | | | | |
Collapse
|
|
15
|
Bulynko YA, O'Malley BW. Nuclear receptor coactivators: structural and functional biochemistry. Biochemistry 2010; 50:313-28. [PMID: 21141906 DOI: 10.1021/bi101762x] [Citation(s) in RCA: 77] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Transcription of eukaryotic cell is a multistep process tightly controlled by concerted action of macromolecules. Nuclear receptors are ligand-activated sequence-specific transcription factors that bind DNA and activate (or repress) transcription of specific sets of nuclear target genes. Successful activation of transcription by nuclear receptors and most other transcription factors requires "coregulators" of transcription. Coregulators make up a diverse family of proteins that physically interact with and modulate the activity of transcription factors and other components of the gene expression machinery via multiple biochemical mechanisms. The coregulators include coactivators that accomplish reactions required for activation of transcription and corepressors that suppress transcription. This review summarizes our current knowledge of nuclear receptor coactivators with an emphasis on their biochemical mechanisms of action and means of regulation.
Collapse
Affiliation(s)
- Yaroslava A Bulynko
- Molecular and Cellular Biology, BCM130 Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
| | | |
Collapse
|
|
16
|
Abstract
Paraspeckles are a relatively new class of subnuclear bodies found in the interchromatin space of mammalian cells. They are RNA-protein structures formed by the interaction between a long nonprotein-coding RNA species, NEAT1/Men epsilon/beta, and members of the DBHS (Drosophila Behavior Human Splicing) family of proteins: P54NRB/NONO, PSPC1, and PSF/SFPQ. Paraspeckles are critical to the control of gene expression through the nuclear retention of RNA containing double-stranded RNA regions that have been subject to adenosine-to-inosine editing. Through this mechanism paraspeckles and their components may ultimately have a role in controlling gene expression during many cellular processes including differentiation, viral infection, and stress responses.
Collapse
Affiliation(s)
- Archa H Fox
- Western Australian Institute for Medical Research and Centre For Medical Research, University of Western Australia, Crawley, 6009 Western Australia, Australia.
| | | |
Collapse
|
|
17
|
Apostolatos H, Apostolatos A, Vickers T, Watson JE, Song S, Vale F, Cooper DR, Sanchez-Ramos J, Patel NA. Vitamin A metabolite, all-trans-retinoic acid, mediates alternative splicing of protein kinase C deltaVIII (PKCdeltaVIII) isoform via splicing factor SC35. J Biol Chem 2010; 285:25987-95. [PMID: 20547768 DOI: 10.1074/jbc.m110.100735] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Vitamin A metabolite, all-trans-retinoic acid (RA), induces cell growth, differentiation, and apoptosis and has an emerging role in gene regulation and alternative splicing events. Protein kinase Cdelta (PKCdelta), a serine/threonine kinase, has a role in cell proliferation, differentiation, and apoptosis. We reported an alternatively spliced variant of human PKCdelta, PKCdeltaVIII that functions as a pro-survival protein (1). RA regulates the splicing and expression of PKCdeltaVIII via utilization of a downstream 5' splice site of exon 10 on PKCdelta pre-mRNA. Here, we further elucidate the molecular mechanisms involved in RA regulation of alternative splicing of PKCdeltaVIII mRNA. Overexpression and knockdown of the splicing factor SC35 (i.e. SRp30b) indicated that it is involved in PKCdeltaVIII alternative splicing. To identify the cis-elements involved in 5' splice site selection we cloned a minigene, which included PKCdelta exon 10 and its flanking introns in the pSPL3 splicing vector. Alternative 5' splice site utilization in the minigene was promoted by RA. Further, co-transfection of SC35 with PKCdelta minigene promoted selection of 5' splice site II. Mutation of the SC35 binding site in the PKCdelta minigene abolished RA-mediated utilization of 5' splice splice II. RNA binding assays demonstrated that the enhancer element downstream of PKCdelta exon 10 is a SC35 cis-element. We conclude that SC35 is pivotal in RA-mediated PKCdelta pre-mRNA alternative splicing. This study demonstrates how a nutrient, vitamin A, via its metabolite RA, regulates alternative splicing and thereby gene expression of the pro-survival protein PKCdeltaVIII.
Collapse
|
|
18
|
Alternative splicing in stem cell self-renewal and diferentiation. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2010; 695:92-104. [PMID: 21222201 DOI: 10.1007/978-1-4419-7037-4_7] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
This chapter provides a review of recent advances in understanding the importance of alternative pre-messenger RNA splicing in stem cell biology. The majority of transcribed pre-mRNAs undergo RNA splicing where introns are excised and exons are juxtaposed to form mature messenger RNA sequences. This regulated, selective removal of whole or portions of exons by alternative splicing provides avenues for control of RNA abundance and proteome diversity. We discuss several examples of key alternative splicing events in stem cell biology and provide an overview of recently developed microarray and sequencing technologies that enable systematic and genome-wide assessment of the extent of alternative splicing during stem cell differentiation.
Collapse
|
|
19
|
Fu RH, Liu SP, Ou CW, Yu HH, Li KW, Tsai CH, Shyu WC, Lin SZ. Alternative Splicing Modulates Stem Cell Differentiation. Cell Transplant 2009; 18:1029-38. [PMID: 19523332 DOI: 10.3727/096368909x471260] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Stem cells have the surprising potential to develop into many different cell types. Therefore, major research efforts have focused on transplantation of stem cells and/or derived progenitors for restoring depleted diseased cells in degenerative disorders. Understanding the molecular controls, including alternative splicing, that arise during lineage differentiation of stem cells is crucial for developing stem cell therapeutic approaches in regeneration medicine. Alternative splicing to allow a single gene to encode multiple transcripts with different protein coding sequences and RNA regulatory elements increases genomic complexities. Utilizing differences in alternative splicing as a molecular marker may be more sensitive than simply gene expression in various degrees of stem cell differentiation. Moreover, alternative splicing maybe provide a new concept to acquire induced pluripotent stem cells or promote cell–cell transdifferentiation for restorative therapies and basic medicine researches. In this review, we highlight the recent advances of alternative splicing regulation in stem cells and their progenitors. It will hopefully provide much needed knowledge into realizing stem cell biology and related applications.
Collapse
Affiliation(s)
- Ru-Huei Fu
- Center for Neuropsychiatry, China Medical University Hospital, Taichung, Taiwan
- Graduate Institute of Immunology, China Medical University, Taichung, Taiwan
| | - Shih-Ping Liu
- Center for Neuropsychiatry, China Medical University Hospital, Taichung, Taiwan
- Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan
| | - Chen-Wei Ou
- Graduate Institute of Immunology, China Medical University, Taichung, Taiwan
| | - Hsiu-Hui Yu
- Center for Neuropsychiatry, China Medical University Hospital, Taichung, Taiwan
| | - Kuo-Wei Li
- Center for Neuropsychiatry, China Medical University Hospital, Taichung, Taiwan
| | - Chang-Hai Tsai
- Department of Pediatrics, China Medical University Hospital, Taichung, Taiwan
- Department of Healthcare Administration, Asia University, Taichung, Taiwan
| | - Woei-Cherng Shyu
- Center for Neuropsychiatry, China Medical University Hospital, Taichung, Taiwan
- Graduate Institute of Immunology, China Medical University, Taichung, Taiwan
| | - Shinn-Zong Lin
- Center for Neuropsychiatry, China Medical University Hospital, Taichung, Taiwan
- Graduate Institute of Immunology, China Medical University, Taichung, Taiwan
- China Medical University Beigang Hospital, Yunlin, Taiwan
| |
Collapse
|
|
20
|
Brooks YS, Wang G, Yang Z, Smith KK, Bieberich E, Ko L. Functional pre- mRNA trans-splicing of coactivator CoAA and corepressor RBM4 during stem/progenitor cell differentiation. J Biol Chem 2009; 284:18033-46. [PMID: 19416963 PMCID: PMC2709364 DOI: 10.1074/jbc.m109.006999] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2009] [Revised: 04/10/2009] [Indexed: 01/23/2023] Open
Abstract
Alternative splicing yields functionally distinctive gene products, and their balance plays critical roles in cell differentiation and development. We have previously shown that tumor-associated enhancer loss in coactivator gene CoAA leads to its altered alternative splicing. Here we identified two intergenic splicing variants, a zinc finger-containing coactivator CoAZ and a non-coding transcript ncCoAZ, between CoAA and its downstream corepressor gene RBM4. During stem/progenitor cell neural differentiation, we found that the switched alternative splicing and trans-splicing between CoAA and RBM4 transcripts result in lineage-specific expression of wild type CoAA, RBM4, and their variants. Stable expression of CoAA, RBM4, or their variants prevents the switch and disrupts the embryoid body formation. In addition, CoAA and RBM4 counter-regulate the target gene Tau at exon 10, and their splicing activities are subjected to the control by each splice variant. Further phylogenetic analysis showed that mammalian CoAA and RBM4 genes share common ancestry with the Drosophila melanogaster gene Lark, which is known to regulate early development and circadian rhythms. Thus, the trans-splicing between CoAA and RBM4 transcripts may represent a required regulation preserved during evolution. Our results demonstrate that a linked splicing control of transcriptional coactivator and corepressor is involved in stem/progenitor cell differentiation. The alternative splicing imbalance of CoAA and RBM4, because of loss of their common enhancer in cancer, may deregulate stem/progenitor cell differentiation.
Collapse
Affiliation(s)
- Yang S. Brooks
- From the Institute of Molecular Medicine and Genetics and
| | - Guanghu Wang
- From the Institute of Molecular Medicine and Genetics and
| | - Zheqiong Yang
- From the Institute of Molecular Medicine and Genetics and
| | - Kimberly K. Smith
- Department of Pathology, Medical College of Georgia, Augusta, Georgia 30912
| | | | - Lan Ko
- From the Institute of Molecular Medicine and Genetics and
- Department of Pathology, Medical College of Georgia, Augusta, Georgia 30912
| |
Collapse
|
|
21
|
Ohe K, Tamai KT, Parvinen M, Sassone-Corsi P. DAX-1 and SOX6 molecular interplay results in an antagonistic effect in pre-mRNA splicing. Dev Dyn 2009; 238:1595-604. [DOI: 10.1002/dvdy.21957] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
|
|
22
|
Casado P, Prado MA, Zuazua-Villar P, Del Valle E, Artime N, Cabal-Hierro L, Rupérez P, Burlingame AL, Lazo PS, Ramos S. Microtubule interfering agents and KSP inhibitors induce the phosphorylation of the nuclear protein p54(nrb), an event linked to G2/M arrest. J Proteomics 2008; 71:592-600. [PMID: 18832053 DOI: 10.1016/j.jprot.2008.09.001] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2008] [Revised: 08/19/2008] [Accepted: 09/08/2008] [Indexed: 01/19/2023]
Abstract
Microtubule interfering agents (MIAs) are anti-tumor drugs that inhibit microtubule dynamics, while kinesin spindle protein (KSP) inhibitors are substances that block the formation of the bipolar spindle during mitosis. All these compounds cause G2/M arrest and cell death. Using 2D-PAGE followed by Nano-LC-ESI-Q-ToF analysis, we found that MIAs such as vincristine (Oncovin) or paclitaxel (Taxol) and KSP inhibitors such as S-tritil-l-cysteine induce the phosphorylation of the nuclear protein p54(nrb) in HeLa cells. Furthermore, we demonstrate that cisplatin (Platinol), an anti-tumor drug that does not cause M arrest, does not induce this modification. We show that the G2/M arrest induced by MIAs is required for p54(nrb) phosphorylation. Finally, we demonstrate that CDK activity is required for MIA-induced phosphorylation of p54(nrb).
Collapse
Affiliation(s)
- Pedro Casado
- Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Oncología del Principado de Asturias, Universidad de Oviedo, 33071, Oviedo, Spain
| | | | | | | | | | | | | | | | | | | |
Collapse
|
|
23
|
Conway-Campbell BL, Brooks AJ, Robinson PJ, Perani M, Waters MJ. The extracellular domain of the growth hormone receptor interacts with coactivator activator to promote cell proliferation. Mol Endocrinol 2008; 22:2190-202. [PMID: 18635665 PMCID: PMC5419461 DOI: 10.1210/me.2008-0128] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2008] [Accepted: 07/07/2008] [Indexed: 02/08/2023] Open
Abstract
The presence of GH receptor (GHR) in the cell nucleus correlates with cell division, and targeting the GHR to the nucleus results in constitutive proliferation and transformation because of increased sensitivity to autocrine GH. Here we have sought additional mechanisms that might account for the enhanced proliferation seen with nuclear GHR, commencing with a yeast two-hybrid (Y2H) screen for interactors with the extracellular domain of the GHR [GH-binding protein (GHBP)]. We find that the GHBP is a transcriptional activator in yeast and mammalian cells, and this activity resides in the lower cytokine receptor module. Activity is dependent on S226, the conserved serine of the cytokine receptor consensus WSXWS box. By using parallel GHBP affinity columns and tandem mass spectrometry of tryptic digests of proteins bound to wild-type GHBP and S226A columns, we identified proteins that bind to the transcriptionally active GHBP. These include a nucleoporin and two transcriptional regulators, notably the coactivator activator (CoAA), which is also an RNA binding splicing protein. Binding of CoAA to the GHBP was confirmed by glutathione S-transferase pulldown and coimmunoprecipitation, and shown to be GH dependent in pro-B Ba/F3 cells. Importantly, stable expression of CoAA in Ba/F3 cells resulted in an increased maximum proliferation in response to GH, but not IL-3. Because CoAA overexpression has been identified in many cancers and its stable expression promotes cell proliferation and cell transformation in NIH-3T3 cells, we suggest CoAA contributes to the proliferative actions of nuclear GHR by the hormone-dependent recruitment of this powerful coactivator to the GHR.
Collapse
|