Nat Cell Biol
May 1, 2009;
Arginine methylation of Piwi proteins catalysed by dPRMT5 is required for Ago3 and Aub stability.
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
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
Nat Cell Biol
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Figure 2. Xenopus laevis Piwi proteins with bound piRNAs are immunoprecipitated by Y12 and contain sDMAs(a) Protein immunoprecipitates from indicated X. laevis tissues; Xili and Xiwi were identified by mass spectrometry (Supplementary Table 3).(b) Immunoprecipitates from X. laevis oocytes were probed on Western blots with indicated antibodies. Band with asterisk is bovine IgG from tissue culture supernatant of anti-Mili hybridoma.(c) RNA-immunoprecipitations from X. laevis.(d) Periodate oxidation and β-elimination of X. laevis piRNAs isolated from Y12 immunoprecipitates.(e) Nucleotide composition of X. laevis piRNAs.(f) Northern blot for XL-piR-3(g) In situ hybridization for XL-piR-3 in X. laevis oocyte; bar = 100μm
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
Valois, a component of the nuage and pole plasm, is involved in assembly of these structures, and binds to Tudor and the methyltransferase Capsuléen.