Arginine methylation of Aubergine mediates Tudor binding and germ plasm localization

Abstract

Piwi proteins such as Drosophila Aubergine (Aub) and mouse Miwi are essential for germline development and for primordial germ cell (PGC) specification. They bind piRNAs and contain symmetrically dimethylated arginines (sDMAs), catalyzed by dPRMT5. PGC specification in Drosophila requires maternal inheritance of cytoplasmic factors, including Aub, dPRMT5, and Tudor (Tud), that are concentrated in the germ plasm at the posterior end of the oocyte. Here we show that Miwi binds to Tdrd6 and Aub binds to Tudor, in an sDMA-dependent manner, demonstrating that binding of sDMA-modified Piwi proteins with Tudor-domain proteins is an evolutionarily conserved interaction in germ cells. We report that in Drosophila tud(1) mutants, the piRNA pathway is intact and most transposons are not de-repressed. However, the localization of Aub in the germ plasm is severely reduced. These findings indicate that germ plasm assembly requires sDMA modification of Aub by dPRMT5, which, in turn, is required for binding to Tudor. Our study also suggests that the function of the piRNA pathway in PGC specification may be independent of its role in transposon control.

FIGURE 1.

Drosophila Tudor protein and its mouse homolog, Tdrd6, bind specifically to sDMAs of peptides derived from Aub and Miwi proteins, respectively. (A) Biotinylated peptides used for pull-downs. (B) Mouse testis or (C) Drosophila ovary lysates were incubated with indicated peptides; bound proteins were visualized by silver staining and identified by mass spectrometry. Western blots of indicated proteins from pull-downs are shown.

FIGURE 2.

Miwi interacts with Tdrd6 in vivo, and sDMAs of Aub are required for Tudor binding in vivo. (A) Properties of anti-Miwi antibody. Mouse testis cell lysate and immunoprecipitates (IP) using anti-Miwi or non-immune rabbit serum (NRS, negative control) were probed with anti-Miwi antbody. (B) Tdrd6 or NRS immunoprecipitates from mouse testis were probed with indicated antibodies; total lysates prior to immunoprecipitation are shown. (C) Tudor or NRS immunoprecipitates from wild-type (wt) and dPRMT5-null ovaries were probed with indicated antibodies.

FIGURE 3.

sDMAs of Aub are required for binding to Tudor in vitro. (A) Sequences of Aub-WT and mutant Aub-4K, showing the four arginines that are substrates for methylation and that were substituted for lysines. (B) In vitro translated and radiolabeled full-length Tudor was incubated with immunopurified Flag-tagged wild-type Aub (wt) or mutant Aub (Aub-4K, not containing sDMAs) produced is S2 cells, or with Flag-beads only. Bound proteins were analyzed by NuPAGE and visualized by autoradiography. Input shows 10% of in vitro translated Tudor. Amounts of Aub-wt and Aub-4K were analyzed by Western blot using Flag antibody. (C) Schematic of domain architecture and deletion mutants of Tudor; (boxes) individual Tudor domains; (white boxes) deleted domains. Ability to support germ cell formation is indicated (Arkov et al. 2006). (D) In vitro translated and radiolabeled deletion mutants of Tudor were incubated with immunopurified Aub-wt or Aub-4K, or with Flag-beads only. Bound proteins were analyzed by NuPAGE and visualized by autoradiography. Input shows 10% of in vitro translated Tudor deletion mutants. (E) Amounts of Aub-wt and Aub-4K of each binding reaction were analyzed by Western blot using Flag antibody.

FIGURE 4.

Tudor is essential for localization of Aub to the pole plasm but does not affect the levels of Piwi proteins or piRNAs or of most transposon transcripts. (A) Western blots of Drosophila ovary lysates from wild-type (WT) or tudor (tud¹) with indicated antibodies. (B) Aub or Piwi immunoprecipitates from ovary lysates were probed with Aub and Piwi antibodies, and (C) bound piRNAs were 5′-end-radiolabeled and analyzed by UREA-PAGE. (D) Levels of indicated transposon transcripts in ovaries of wild-type, tud¹, and csul/dPRMT5-null were examined by qRT-PCR. (Red line) Transcript levels from wild-type ovaries were set as 1, and fold-changes are indicated; averages of three independent experiments with SD values are shown. Localization of Aub in wt and tud¹ ovaries in (E) early or (F) late egg chambers; (arrow) pole (germ) plasm.

FIGURE 5.

Model depicting interactions of indicated proteins in pole (germ) plasm formation.