Alternative Splicing of OsRAD1 Defines C-Terminal Domain Essential for Protein Function in Meiosis
#These authors contributed equally to this work
Received date: 2019-08-17
Accepted date: 2019-10-21
Online published: 2020-03-31
Alternative splicing can generate multiple mRNAs that differ in their untranslated regions or coding sequences, and these differences might affect mRNA stability or result in different protein isoforms with diverse functions and/or localizations. In this study, we isolated a sterile mutant in rice with abnormal meiosis of microspore mother cells and megaspore mother cells that carried a point mutation in OsRAD1 gene. Cloning of OsRAD1 cDNAs revealed three transcript variants, named as OsRAD1.1, OsRAD1.2 and OsRAD1.3, respectively, which were derived from alternative splicing of the last intron. Proteins derived from the three transcripts were mostly identical except the difference in the very C-terminal domain. The three transcripts exhibited similar expression patterns in various tissues, but the expression level of OsRAD1.1 was the highest. Specific knockout of OsRAD1.1 led to sterility, while knockout of OsRAD1.2 and OsRAD1.3 together did not change the plant fertility. Overexpression of OsRAD1.2 and OsRAD1.3 cDNAs in OsRAD1.1-specific mutant did not complement the plant fertility. Yeast two-hybrid assay showed that OsRAD1.1, but not OsRAD1.2 and OsRAD1.3, interacted with the three other meiosis proteins OsHUS1, OsRAD9 and OsRAD17, suggesting that the C-terminal domain of OsRAD1.1 is critical for the protein function.
Key words： Oryza sativa; OsRAD1; alternative splicing; meiosis; protein interaction; fertility; sterility
Shuting Yuan, Chunjue Xu, Wei Yan, Zhenyi Chang, Xingwang Deng, Zhufeng Chen, Jianxin Wu, Xiaoyan Tang . Alternative Splicing of OsRAD1 Defines C-Terminal Domain Essential for Protein Function in Meiosis[J]. Rice Science, 2020 , 27(4) : 289 -301 . DOI: 10.1016/j.rsci.2020.05.005
| [1] | Bermudez V P, Lindsey-Boltz L A, Cesare A J, Maniwa Y, Griffith J D, Hurwitz J, Sancar A. 2003. Loading of the human 9-1-1 checkpoint complex onto DNA by the checkpoint clamp loader hRad17-replication factor C complex in vitro. Proc Natl Acad Sci USA, 100(4): 1633-1638. |
| [2] | Black D L. 2003. Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem, 72(1): 291-336. |
| [3] | Chang Z Y, Chen Z F, Wang N, Xie G, Lu J W, Yan W, Zhou J L, Tang X Y, Deng X W. 2016. Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility gene. Proc Natl Acad Sci USA, 113(49): 14145-14150. |
| [4] | Chaudhary S, Jabre I, Reddy A S N, Staiger D, Syed N H. 2019. Perspective on alternative splicing and proteome complexity in plants. Trends Plant Sci, 24(6): 496-506. |
| [5] | Che L X, Wang K J, Tang D, Liu Q Q, Chen X J, Li Y F, Hu Q, Shen Y, Yu H X, Gu M H, Cheng Z K. 2014. OsHUS1 facilitates accurate meiotic recombination in rice. PLoS Genet, 10(6): e1004405. |
| [6] | Chen M, Manley J L. 2009. Mechanisms of alternative splicing regulation: Insights from molecular and genomics approaches. Nat Rev Mol Cell Biol, 10(11): 741-754. |
| [7] | Chen Z F, Lu J W, Lu Q Q, Wang N, Wang C X, Xie G, Zhou X Y, Tang X Y. 2014. Screening and analysis of male sterile mutants derived from elite indica cultivar Huanghuazhan. Guangdong Agric Sci, 41(19): 1-4. (in Chinese) |
| [8] | Doré A S, Kilkenny M L, Rzechorzek N J, Pearl L H. 2009. Crystal structure of the Rad9-Rad1-Hus1 DNA damage checkpoint complex-implications for clamp loading and regulation. Mol Cell, 34(6): 735-745. |
| [9] | Freire R, Murguía J R, Tarsounas M, Lowndes N F, Moens P B, Jackson S P. 1998. Human and mouse homologs of Schizosa- ccharomyces pombe rad1+ and Saccharomyces cerevisiae RAD17: Linkage to checkpoint control and mammalian meiosis. Genes Dev, 12(16): 2560-2573. |
| [10] | Griffith J D, Lindsey-Boltz L A, Sancar A. 2002. Structures of the human Rad17-replication factor C and checkpoint Rad 9-1-1 complexes visualized by glycerol spray/low voltage microscopy. J Biol Chem, 277(18): 15233-15236. |
| [11] | Grushcow J M, Holzen T M, Park K J, Weinert T, Lichten M, Bishop D K. 1999. Saccharomyces cerevisiae checkpoint genes MEC1, RAD17 and RAD24 are required for normal meiotic recombination partner choice. Genetics, 153(2): 607-620. |
| [12] | Han L, Hu Z S, Liu Y H, Wang X Y, Hopkins K M, Lieberman H B, Hang H Y. 2010. Mouse Rad1 deletion enhances susceptibility for skin tumor development. Mol Cancer, 9(1): 67. |
| [13] | Heitzeberg F, Chen I P, Hartung F, Orel N, Angelis K J, Puchta H. 2004. The Rad17 homologue of Arabidopsis is involved in the regulation of DNA damage repair and homologous recombination. Plant J, 38(6): 954-968. |
| [14] | Hu Q, Tang D, Wang H J, Shen Y, Chen X J, Ji J H, Du G J, Li Y F, Cheng Z K. 2016. The exonuclease homolog OsRAD1 promotes accurate meiotic double-strand break repair by suppressing nonhomologous end joining. Plant Physiol, 172(2): 1105-1116. |
| [15] | Hu Q, Zhang C, Xue Z H, Ma L J, Liu W, Shen Y, Ma B J, Cheng Z K. 2018. OsRAD17 is required for meiotic double-strand break repair and plays a redundant role with OsZIP4 in synaptonemal complex assembly. Front Plant Sci, 9: 1236. |
| [16] | Jaramillo-Lambert A, Harigaya Y, Vitt J, Villeneuve A, Engebrecht J. 2010. Meiotic errors activate checkpoints that improve gamete quality without triggering apoptosis in male germ cells. Curr Biol, 20(23): 2078-2089. |
| [17] | Kalsotra A, Cooper T A. 2011. Functional consequences of developmentally regulated alternative splicing. Nat Rev Genet, 12(10): 715-729. |
| [18] | Kelemen O, Convertini P, Zhang Z Y, Wen Y, Shen M, Falaleeva M, Stamm S. 2013. Function of alternative splicing. Gene, 514(1): 1-30. |
| [19] | Laloum T, Martín G, Duque P. 2018. Alternative splicing control of abiotic stress responses. Trends Plant Sci, 23(2): 140-150. |
| [20] | Lochlainn S Ó, Amoah S, Graham N S, Alamer K, Rios J J, Kurup S, Stoute A, Hammond J P, Østergaard L, King G J, White P J, Broadley M R. 2011. High resolution melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes. Plant Methods, 7(1): 43. |
| [21] | Luo Q, Li Y F, Shen Y, Cheng Z K. 2014. Ten years of gene discovery for meiotic event control in rice. J Genet Genom, 41(3): 125-137. |
| [22] | Lyndaker A M, Lim P X, Mleczko J M, Diggins C E, Holloway J K, Holmes R J, Kan R, Schlafer D H, Freire R, Cohen P E, Weiss R S. 2013. Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance. PLoS Genet, 9(2): e1003320. |
| [23] | Ma X L, Zhang Q Y, Zhu Q L, Liu W, Chen Y, Qiu R, Wang B, Yang Z F, Li H Y, Lin Y R, Xie Y Y, Shen R X, Chen S F, Wang Z, Chen Y L, Guo J X, Chen L T, Zhao X C, Dong Z C, Liu Y G. 2015. A robust CRISPR/Cas9 system for convenient, high- efficiency multiplex genome editing in monocot and dicot plants. Mol Plant, 8(8): 1274-1284. |
| [24] | Melo J, Toczyski D. 2002. A unified view of the DNA-damage checkpoint. Curr Opin Cell Biol, 14(2): 237-245. |
| [25] | Naftelberg S, Schor I E, Ast G, Kornblihtt A R. 2015. Regulation of alternative splicing through coupling with transcription and chromatin structure. Annu Rev Biochem, 84(1): 165-198. |
| [26] | Navadgi-Patil V M, Burgers P M. 2009. A tale of two tails: Activation of DNA damage checkpoint kinase Mec1/ATR by the 9-1-1 clamp and by Dpb11/TopBP1. DNA Repair, 8(9): 996-1003. |
| [27] | Nonomura K, Nakano M, Fukuda T, Eiguchi M, Miyao A, Hirochika H, Kurata N. 2004. The novel gene HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1 of rice encodes a putative coiled-coil protein required for homologous chromosome pairing in meiosis. Plant Cell, 16(4): 1008-1020. |
| [28] | Nonomura K, Nakano M, Eiquchi M, Suzuki T, Kurata N. 2006. PAIR2 is essential for homologous chromosome synapsis in rice meiosis I. J Cell Sci, 119(2): 217-225. |
| [29] | Nonomura K, Morohoshi A, Nakano M, Eiguchi M, Miyao A, Hirochika H, Kurata N. 2007. A germ cell specific gene of the ARGONAUTE family is essential for the progression of premeiotic mitosis and meiosis during sporogenesis in rice. Plant Cell, 19(8): 2583-2594. |
| [30] | Ohashi E, Tsurimoto T. 2017. Functions of multiple clamp and clamp-loader complexes in eukaryotic DNA replication. Adv Exp Med Biol, 1042: 135-162. |
| [31] | Pan Q, Shai O, Lee L J, Frey B J, Blencowe B J. 2008. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet, 40(12): 1413-1415. |
| [32] | Peretz G, Arie L G, Bakhrat A, Abdu U. 2009. The Drosophila hus1 gene is required for homologous recombination repair during meiosis. Mech Dev, 126: 677-686. |
| [33] | Shinohara M, Sakai K, Ogawa T, Shinohara A. 2003. The mitotic DNA damage checkpoint proteins Rad17 and Rad24 are required for repair of double-strand breaks during meiosis in yeast. Genetics, 164(3): 855-865. |
| [34] | Szakonyi D, Duque P. 2018. Alternative splicing as a regulator of early plant development. Front Plant Sci, 9: 1174. |
| [35] | Udell C M, Lee S K, Davey S. 1998. HRAD1 and MRAD1 encode mammalian homologues of the fission yeast rad1+ cell cycle checkpoint control gene. Nucl Acids Res, 26(17): 3971-3976. |
| [36] | Uri A, Martha K, Veronika B I, Anna B, Trudi S. 2007. An essential role for Drosophila hus1 in somatic and meiotic DNA damage responses. J Cell Sci, 120(6): 1042-1049. |
| [37] | Vasileva A, Hopkins K M, Wang X Y, Weissbach M M, Friedman R A, Wolgemuth D J, Lieberman H B. 2013. The DNA damage checkpoint protein RAD9A is essential for male meiosis in the mouse. J Cell Sci, 126(17): 3927-3938. |
| [38] | Venclovas C, Thelen M P. 2000. Structure-based predictions of Rad1, Rad9, Hus1 and Rad17 participation in sliding clamp and clamp-loading complexes. Nucl Acids Res, 28(13): 2481-2493. |
| [39] | Wang C L, Wang Y, Cheng Z J, Zhao Z G, Chen J, Sheng P K, Yu Y, Ma W W, Duan E C, Wu F Q, Liu L L, Qin R Z, Zhang X, Guo X P, Wang J L, Jiang L, Wan J M. 2016. The role of OsMSH4 in male and female gamete development in rice meiosis. J Exp Bot, 67(5): 1447-1459. |
| [40] | Wang M, Wang K J, Tang D, Wei C X, Li M, Shen Y, Chi Z C, Gu M H, Cheng Z K. 2010. The central element protein ZEP1 of the synaptonemal complex regulates the number of crossovers during meiosis in rice. Plant Cell, 22(2): 417-430. |
| [41] | Wang Y, Copenhaver G P. 2018. Meiotic recombination: Mixing it up in plants. Annu Rev Plant Biol, 69: 577-609. |
| [42] | Xu M, Bai L, Gong Y, Xie W, Hang H Y, Jiang T. 2009. Structure and functional implications of the human Rad9-Hus1-Rad1 cell cycle checkpoint complex. J Biol Chem, 284(31): 20457-20461. |
| [43] | Yan W, Chen Z F, Lu J W, Xu C J, Xie G, Li Y Q, Deng X W, He H, Tang X Y. 2017. Simultaneous identification of multiple causal mutations in rice. Front Plant Sci, 7: 2055. |
| [44] | Yuan W Y, Li X W, Chang Y X, Wen R Y, Chen G X, Zhang Q F, Wu C Y. 2009. Mutation of the rice gene PAIR3 results in lack of bivalent formation in meiosis. Plant J, 59(2): 303-315. |
| [45] | Zhang C B, Liu Y H, Hu Z S, An L L, He Y K, Hang H Y. 2011. Targeted deletion of mouse Rad1 leads to deficient cellular DNA damage responses. Protein Cell, 2(5): 410-422. |
| [46] | Zhang D B, Luo X, Zhu L. 2011. Cytological analysis and genetic control of rice anther development. J Genet Genom, 38(9): 379-390. |
| [47] | Zhou B B, Elledge S J. 2000. The DNA damage response: Putting checkpoints in perspective. Nature, 408: 433-439. |
| [48] | Zou L, Cortez D, Elledge S J. 2002. Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin. Genes Dev, 16(2): 198-208. |
/
| 〈 |
|
〉 |