XB-ART-39542Nat Cell Biol May 1, 2009; 11 (5): 652-8.
Piwi family proteins are essential for germline development and bind piwi-interacting RNAs (piRNAs). The grandchildless gene aub of Drosophila melanogaster encodes the piRNA-binding protein Aubergine (Aub), which is essential for formation of primordial germ cells (PGCs). Here we report that Piwi family proteins of mouse, Xenopus laevis and Drosophila contain symmetrical dimethylarginines (sDMAs). We found that Piwi proteins are expressed in Xenopus oocytes and we identified numerous Xenopus piRNAs. We report that the Drosophila homologue of protein methyltransferase 5 (dPRMT5, csul/dart5), which is also the product of a grandchildless gene, is required for arginine methylation of Drosophila Piwi, Ago3 and Aub proteins in vivo. Loss of dPRMT5 activity led to a reduction in the levels of piRNAs, Ago3 and Aub proteins, and accumulation of retrotransposons in the Drosophila ovary. Our studies explain the relationship between aub and dPRMT5 (csul/dart5) genes by demonstrating that dPRMT5 is the enzyme that methylates Aub. Our findings underscore the significance of sDMA modification of Piwi proteins in the germline and suggest an interacting pathway of genes that are required for piRNA function and PGC specification.
PubMed ID: 19377467
PMC ID: PMC2746449
Article link: Nat Cell Biol
Grant support: GM0720777 NIGMS NIH HHS , GM76621 NIGMS NIH HHS , NS046573 NINDS NIH HHS , NS056070 NINDS NIH HHS , R01 NS046573-04 NINDS NIH HHS , UL1RR024134 NCRR NIH HHS , R01 GM076621-01A1 NIGMS NIH HHS , R01 GM076621-02 NIGMS NIH HHS , R01 GM076621-03 NIGMS NIH HHS , R01 GM076621 NIGMS NIH HHS , R01 NS046573 NINDS NIH HHS , R01 NS056070 NINDS NIH HHS , UL1 RR024134 NCRR NIH HHS
Genes referenced: ago3 pgc pir piwil1 piwil2 prmt5 slc22a18
Article Images: [+] show captions
|Figure 3. Drosophila PRMT5 (csul, dart5) is required for arginine methylation of Aub, Piwi and Ago3 proteins in ovaries(a) Western blots from wild-type (WT) or csul (dPRMT5) mutant (−/−) ovary. Piwi or Aub immunoprecipitates from ovary lysates were probed on western blots with anti-Piwi and anti-Aub antibody(b); or SYM11 and ASYM24 (c).(d) Ago3 immunoprecipitates from WT or csul mutant (−/−) ovary lysates were probed on Western blots (WB) with indicated antibodies.(e) Sequences of wild-type (WT) and mutant (M) Aub, where the four arginines that are substrates for methylation were substituted with lysines.(f) Anti-Flag immunoprecipitates of S2 cells stably transfected with WT or M Flag-Aub were probed on western blots with indicated antibodies.(g) Crosslinking of synthetic, radiolabeled piRNA to immunopurified WT or M Flag-Aub.(h) RNA immunoprecipitation.(i) Periodate oxidation and β-elimination of Drosophila piRNAs isolated from Piwi immunoprecipitates from wt or csul (−/−) mutant ovaries.|
|Figure 4. Reduction of Piwi proteins and piRNAs with accumulation of HeT-A retrotransposons and marked reduction of the Aub protein that localizes in the pole plasm, in the absence of dPRMT5 (csul) activity(a) Western blots of Drosophila ovary lysates from wild-type (wt), heterozygous (+/−) or homozygous (−/−) csul.(b) Northern blots of total RNA from indicated ovary lysates.(c) Fold changes of HeT-A retrotransposon transcripts in indicated ovaries assessed with qRT-PCR; n=3 and s.d. shown.(d) Ago 3, Aub and Piwi localization in indicated early stage egg chambers; bar = 15μm(e) Aub localization in indicated csul oocytes. Arrow indicates pole (germ) plasm; bar = 15μm|
|Figure 5. Proposed classification for selected Drosophila melanogaster grandchildless genes|