Piwi proteins associate with piRNAs and functions in epigenetic programming, post-transcriptional regulation, transposon silencing, and germline development. However, it is not known whether the diverse functions of these proteins are molecularly separable. Here we report that Piwi interacts with Tudor-SN (Tudor staphylococcal nuclease, TSN) antagonistically in regulating spermatogenesis but synergistically in silencing transposons. However, it is not required for piRNA biogenesis. TSN is known to participate in diverse molecular functions such as RNAi, degradation of hyper-edited miRNAs, and spliceosome assembly. We show that TSN colocalizes with Piwi in primordial germ cells (PGCs) and embryonic somatic cells. In adult ovaries and testes, TSN is ubiquitously expressed and enriched in the cytoplasm of both germline and somatic cells. The tsn mutants display a higher mitotic index of spermatogonia, accumulation of spermatocytes, defects in meiotic cytokinesis, a decreased number of spermatids, and eventually reduced male fertility. Germline-specific TSN-expression analysis demonstrates that this function is germline-dependent. Different from other known Piwi interters, TSN represses Piwi expression at both protein and mRNA levels. Furthermore, reducing piwi expression in the germline rescues tsn mutant phenotype in a dosage-dependent manner, demonstrating that Piwi and TSN interact antagonistically in germ cells to regulate spermatogenesis. However, the tsn deficiency has little, if any, impact on piRNA biogenesis but displays a synergistic effect with piwi mutants in transposon de-silencing. Our results reveal the biological function of TSN and its contrasting modes of interaction with Piwi in spermatogenesis, transposon silencing, and piRNA biogenesis.
References
[1]
Juliano C, Wang J, Lin H. Uniting germline and stem cells: the function of Piwi proteins and the piRNA pathway in diverse organisms. Annual review of genetics. 2011;45:447–69. doi: 10.1146/annurev-genet-110410-132541. pmid:21942366
[2]
Saxe JP, Lin H. Small noncoding RNAs in the germline. Cold Spring Harbor perspectives in biology. 2011;3(9):a002717. doi: 10.1101/cshperspect.a002717. pmid:21669983
[3]
Watanabe T, Cheng EC, Zhong M, Lin H. Retrotransposons and pseudogenes regulate mRNAs and lncRNAs via the piRNA pathway in the germline. Genome research. 2015;25(3):368–80. doi: 10.1101/gr.180802.114. pmid:25480952
[4]
Peng JC, Lin H. Beyond transposons: the epigenetic and somatic functions of the Piwi-piRNA mechanism. Current opinion in cell biology. 2013;25(2):190–4. doi: 10.1016/j.ceb.2013.01.010. pmid:23465540
[5]
Cox DN, Chao A, Lin H. piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells. Development. 2000;127(3):503–14. pmid:10631171
[6]
Cox DN, Chao A, Baker J, Chang L, Qiao D, Lin H. A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal. Genes & development. 1998;12(23):3715–27. doi: 10.1101/gad.12.23.3715
[7]
Megosh HB, Cox DN, Campbell C, Lin H. The role of PIWI and the miRNA machinery in Drosophila germline determination. Current biology: CB. 2006;16(19):1884–94. pmid:16949822 doi: 10.1016/j.cub.2006.08.051
[8]
Yin H, Lin H. An epigenetic activation role of Piwi and a Piwi-associated piRNA in Drosophila melanogaster. Nature. 2007;450(7167):304–8. Epub 2007/10/24. pmid:17952056 doi: 10.1038/nature06263
[9]
Callebaut I, Mornon JP. The human EBNA-2 coactivator p100: multidomain organization and relationship to the staphylococcal nuclease fold and to the tudor protein involved in Drosophila melanogaster development. Biochem J. 1997;321 (Pt 1):125–32. Epub 1997/01/01. pmid:9003410 doi: 10.1042/bj3210125
[10]
Ponting CP. Tudor domains in proteins that interact with RNA. Trends Biochem Sci. 1997;22(2):51–2. Epub 1997/02/01. pmid:9048482 doi: 10.1016/s0968-0004(96)30049-2
[11]
Shaw N, Zhao M, Cheng C, Xu H, Saarikettu J, Li Y, et al. The multifunctional human p100 protein 'hooks' methylated ligands. Nat Struct Mol Biol. 2007;14(8):779–84. Epub 2007/07/17. pmid:17632523 doi: 10.1038/nsmb1269
[12]
Fashe T, Saarikettu J, Isomaki P, Yang J, Silvennoinen O. Expression analysis of Tudor-SN protein in mouse tissues. Tissue Cell. 2013;45(1):21–31. Epub 2012/10/17. doi: 10.1016/j.tice.2012.09.001. pmid:23068188
[13]
Caudy AA, Ketting RF, Hammond SM, Denli AM, Bathoorn AM, Tops BB, et al. A micrococcal nuclease homologue in RNAi effector complexes. Nature. 2003;425(6956):411–4. Epub 2003/09/26. pmid:14508492 doi: 10.1038/nature01956
[14]
Li CL, Yang WZ, Chen YP, Yuan HS. Structural and functional insights into human Tudor-SN, a key component linking RNA interference and editing. Nucleic Acids Research. 2008;36(11):3579–89. doi: 10.1093/nar/gkn236. pmid:18453631
[15]
Gao X, Zhao X, Zhu Y, He J, Shao J, Su C, et al. Tudor staphylococcal nuclease (Tudor-SN) participates in small ribonucleoprotein (snRNP) assembly via interacting with symmetrically dimethylated Sm proteins. J Biol Chem. 2012;287(22):18130–41. Epub 2012/04/12. doi: 10.1074/jbc.M111.311852. pmid:22493508
[16]
Gao X, Ge L, Shao J, Su C, Zhao H, Saarikettu J, et al. Tudor-SN interacts with and co-localizes with G3BP in stress granules under stress conditions. FEBS Lett. 2010;584(16):3525–32. Epub 2010/07/21. doi: 10.1016/j.febslet.2010.07.022. pmid:20643132
[17]
Scadden AD. The RISC subunit Tudor-SN binds to hyper-edited double-stranded RNA and promotes its cleavage. Nat Struct Mol Biol. 2005;12(6):489–96. Epub 2005/05/17. pmid:15895094 doi: 10.1038/nsmb936
[18]
Yang J, Aittomaki S, Pesu M, Carter K, Saarinen J, Kalkkinen N, et al. Identification of p100 as a coactivator for STAT6 that bridges STAT6 with RNA polymerase II. EMBO J. 2002;21(18):4950–8. Epub 2002/09/18. pmid:12234934 doi: 10.1093/emboj/cdf463
[19]
Paukku K, Yang J, Silvennoinen O. Tudor and nuclease-like domains containing protein p100 function as coactivators for signal transducer and activator of transcription 5. Mol Endocrinol. 2003;17(9):1805–14. Epub 2003/06/24. pmid:12819296 doi: 10.1210/me.2002-0256
[20]
Liu K, Chen C, Guo Y, Lam R, Bian C, Xu C, et al. Structural basis for recognition of arginine methylated Piwi proteins by the extended Tudor domain. Proc Natl Acad Sci U S A. 2010;107(43):18398–403. Epub 2010/10/13. doi: 10.1073/pnas.1013106107. pmid:20937909
[21]
Vagin VV, Wohlschlegel J, Qu J, Jonsson Z, Huang X, Chuma S, et al. Proteomic analysis of murine Piwi proteins reveals a role for arginine methylation in specifying interaction with Tudor family members. Genes Dev. 2009;23(15):1749–62. Epub 2009/07/09. doi: 10.1101/gad.1814809. pmid:19584108
[22]
Liu L, Qi H, Wang J, Lin H. PAPI, a novel TUDOR-domain protein, complexes with AGO3, ME31B and TRAL in the nuage to silence transposition. Development. 2011;138(9):1863–73. doi: 10.1242/dev.059287. pmid:21447556
[23]
Gangaraju VK, Yin H, Weiner MM, Wang JQ, Huang XA, Lin HF. Drosophila Piwi functions in Hsp90-mediated suppression of phenotypic variation. Nature Genetics. 2011;43(2):153–U07. doi: 10.1038/ng.743. pmid:21186352
[24]
Cooper JL, Till BJ, Henikoff S. Fly-TILL: reverse genetics using a living point mutation resource. Fly (Austin). 2008;2(6):300–2. Epub 2008/12/23. doi: 10.4161/fly.7366
[25]
Lin H, Yue L, Spradling AC. The Drosophila fusome, a germline-specific organelle, contains membrane skeletal proteins and functions in cyst formation. Development. 1994;120(4):947–56. Epub 1994/04/01. pmid:7600970
[26]
Yang W, Chendrimada TP, Wang Q, Higuchi M, Seeburg PH, Shiekhattar R, et al. Modulation of microRNA processing and expression through RNA editing by ADAR deaminases. Nat Struct Mol Biol. 2006;13(1):13–21. Epub 2005/12/22. pmid:16369484 doi: 10.1038/nsmb1041
[27]
Cappellari M, Bielli P, Paronetto MP, Ciccosanti F, Fimia GM, Saarikettu J, et al. The transcriptional co-activator SND1 is a novel regulator of alternative splicing in prostate cancer cells. Oncogene. 2013. Epub 2013/09/03. doi: 10.1038/onc.2013.360
[28]
Blanco MA, Aleckovic M, Hua Y, Li T, Wei Y, Xu Z, et al. Identification of staphylococcal nuclease domain-containing 1 (SND1) as a Metadherin-interacting protein with metastasis-promoting functions. J Biol Chem. 2011;286(22):19982–92. Epub 2011/04/12. doi: 10.1074/jbc.M111.240077. pmid:21478147
[29]
Kuruma H, Kamata Y, Takahashi H, Igarashi K, Kimura T, Miki K, et al. Staphylococcal nuclease domain-containing protein 1 as a potential tissue marker for prostate cancer. Am J Pathol. 2009;174(6):2044–50. Epub 2009/05/14. doi: 10.2353/ajpath.2009.080776. pmid:19435788
[30]
Tsuchiya N, Ochiai M, Nakashima K, Ubagai T, Sugimura T, Nakagama H. SND1, a component of RNA-induced silencing complex, is up-regulated in human colon cancers and implicated in early stage colon carcinogenesis. Cancer Res. 2007;67(19):9568–76. Epub 2007/10/03. pmid:17909068 doi: 10.1158/0008-5472.can-06-2707
[31]
Janic A, Mendizabal L, Llamazares S, Rossell D, Gonzalez C. Ectopic expression of germline genes drives malignant brain tumor growth in Drosophila. Science. 2010;330(6012):1824–7. Epub 2011/01/06. doi: 10.1126/science.1195481. pmid:21205669
[32]
Qiao D, Zeeman AM, Deng W, Looijenga LH, Lin H. Molecular characterization of hiwi, a human member of the piwi gene family whose overexpression is correlated to seminomas. Oncogene. 2002;21(25):3988–99. pmid:12037681 doi: 10.1038/sj.onc.1205505
[33]
Ross RJ, Weiner MM, Lin H. PIWI proteins and PIWI-interacting RNAs in the soma. Nature. 2014;505(7483):353–9. doi: 10.1038/nature12987. pmid:24429634
[34]
Saito K, Nishida KM, Mori T, Kawamura Y, Miyoshi K, Nagami T, et al. Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. Genes Dev. 2006;20(16):2214–22. Epub 2006/08/03. pmid:16882972 doi: 10.1101/gad.1454806
[35]
Saarikettu J, Ovod V, Vuoksio M, Gronholm J, Yang J, Silvennoinen O. Monoclonal antibodies against human Tudor-SN. Hybridoma (Larchmt). 2010;29(3):231–6. Epub 2010/06/24. doi: 10.1089/hyb.2009.0114
[36]
Lin H, Spradling AC. A novel group of pumilio mutations affects the asymmetric division of germline stem cells in the Drosophila ovary. Development. 1997;124(12):2463–76. Epub 1997/06/01. pmid:9199372
[37]
Jurka J, Kapitonov VV, Pavlicek A, Klonowski P, Kohany O, Walichiewicz J. Repbase Update, a database of eukaryotic repetitive elements. Cytogenet Genome Res. 2005;110(1–4):462–7. Epub 2005/08/12. pmid:16093699 doi: 10.1159/000084979
[38]
Qi H, Watanabe T, Ku HY, Liu N, Zhong M, Lin H. The Yb Body, a Major Site for Piwi-associated RNA Biogenesis and a Gateway for Piwi Expression and Transport to the Nucleus in Somatic Cells. Journal of Biological Chemistry. 2010;286(5):3789–97. doi: 10.1074/jbc.M110.193888. pmid:21106531
[39]
Saito K, Inagaki S, Mituyama T, Kawamura Y, Ono Y, Sakota E, et al. A regulatory circuit for piwi by the large Maf gene traffic jam in Drosophila. Nature. 2009;461(7268):1296–9. Epub 2009/10/09. doi: 10.1038/nature08501. pmid:19812547
[40]
Malone CD, Brennecke J, Dus M, Stark A, McCombie WR, Sachidanandam R, et al. Specialized piRNA pathways act in germline and somatic tissues of the Drosophila ovary. Cell. 2009;137(3):522–35. Epub 2009/04/28. doi: 10.1016/j.cell.2009.03.040. pmid:19395010
[41]
Lau NC, Robine N, Martin R, Chung WJ, Niki Y, Berezikov E, et al. Abundant primary piRNAs, endo-siRNAs, and microRNAs in a Drosophila ovary cell line. Genome Res. 2009;19(10):1776–85. Epub 2009/06/23. doi: 10.1101/gr.094896.109. pmid:19541914
