Functional involvement of Tudor and dPRMT5 in the piRNA processing pathway in Drosophila germlines

Abstract

In Drosophila, the PIWI proteins, Aubergine (Aub), AGO3, and Piwi are expressed in germlines and function in silencing transposons by associating with PIWI-interacting RNAs (piRNAs). Recent studies show that PIWI proteins contain symmetric dimethyl-arginines (sDMAs) and that dPRMT5/Capsuleen/DART5 is the modifying enzyme. Here, we show that Tudor (Tud), one of Tud domaincontaining proteins, associates with Aub and AGO3, specifically through their sDMA modifications and that these three proteins form heteromeric complexes. piRNA precursor-like molecules are detected in these complexes. The expression levels of Aub and AGO3, along with their degree of sDMA modification, were not changed by tud mutations. However, the population of transposon-derived piRNAs associated with Aub and AGO3 was altered by tud mutations, whereas the total amounts of small RNAs on Aub and AGO3 was increased. Loss of dprmt5 did not change the stability of Aub, but impaired its association with Tud and lowered piRNA association with Aub. Thus, in germline cells, piRNAs are quality-controlled by dPRMT5 that modifies PIWI proteins, in tight association with Tud.

Mitochondrial protein BmPAPI modulates the length of mature piRNAs

“…BmSpn-E depletion resulted in decreased levels of both SIWI-and BmAGO3-bound piRNAs, whereas BmTUDOR depletion increased the levels of the piRNAs. These results are in agreement with previous reports on Drosophila ovaries which implicated Spn-E and TUDOR in piRNA biogenesis, and showed that piRNA levels were severely decreased in spn-E mutant flies and that tudor mutant contained increased piRNA levels (Lim and Kai 2007;Nishida et al 2009). Strikingly, as observed in sDMA-lacking BmAGO3-bound piRNAs, the BmPAPI depletion did not affect quantity but increased the lengths of both SIWI-and BmAGO3-bound piRNAs (Fig.…”

“…The availability of efficient and convenient plasmid transfection and RNAi gene silencing techniques make BmN4 an ideal system to explore the function of TUDOR proteins, and we identified BmPAPI, BmSpn-E, and BmTUDOR as the Bombyx TUDOR proteins responsible for piRNA biogenesis. We were able to recapitulate in BmN4 cells the piRNA behaviors described previously in spn-E and tudor mutant flies (Lim and Kai 2007;Nishida et al 2009): BmSpn-E or BmTUDOR depletion by RNAi resulted in severe decreases or increases in piRNA levels, respectively.…”

“…sDMAs occur in "sDMA motifs" composed of multiple arginine-glycine (RG) and arginine-alanine (RA) repeats in various proteins, and modulate diverse cellular processes. PIWI proteins from various species commonly contain the sDMA motif typically at their N terminus, and evolutionarily conserved presence of PIWI sDMAs has been confirmed in mice, Drosophila, zebrafish, and Xenopus Kirino et al 2009;Nishida et al 2009;Reuter et al 2009;Vagin et al 2009;Huang et al 2011b). The PIWI sDMAs are mediated by PRMT5 methyltransferase, and their functional importance has been demonstrated by the studies on Drosophila null mutants of PRMT5, in which sDMAs are absent in any of the three Drosophila PIWI proteins, leading to transposon activation and defects in germline development (Gonsalvez et al 2006;Anne et al 2007;Kirino et al 2009).…”

Mutations to the piRNA Pathway ComponentAubergineEnhance Meiotic Drive of Segregation Distorter inDrosophila melanogaster

“…In the testes, these two proteins associate with other piRNA pathway components including Vasa (Vas), Armitage (Armi), and Tudor (Tud) in large electron-dense perinuclear bodies, dubbed piNG-bodies, which are thought to regulate the processing and/or targeting of piRNAs to transposable elements via sequence homology (Lim and Kai 2007;Lim et al 2009;Nishida et al 2009;Kibanov et al 2011). The localization of both Aub and AGO3 to this perinuclear region is codependent; loss of one protein through mutation results in mislocalization of the other (Cox et al 2000;Brennecke et al 2007;Nishida et al 2007;Li et al 2009;Nagao et al 2010).…”

Novel mutants of theauberginegene

“…26 The arginine-glycine rich domain (a.k.a. RG domain, aminoacids [11][12][13][14][15][16][17] is necessary to interact with Tudor proteins; 27,28 the PAZ domain (Piwi-ArgonautZwille) located on the N terminal half (aminoacids 280-413) serves as a docking site for 3 0 end of small RNA; 29 and the PIWI domain, located on the C-terminal half (amino acids 554-852) of the protein having a function similar to RNase H 30 ( Fig. 1A).…”