Gender-Specific Hierarchy in Nuage Localization of PIWI-Interacting RNA Factors in Drosophila

Abstract

PIWI-interacting RNAs (piRNAs) are germline-specific small non-coding RNAs that form piRNA-induced silencing complexes (piRISCs) by associating with PIWI proteins, a subclade of the Argonaute proteins predominantly expressed in the germline. piRISCs protect the integrity of the germline genome from invasive transposable DNA elements by silencing them. Multiple piRNA biogenesis factors have been identified in Drosophila. The majority of piRNA factors are localized in the nuage, electron-dense non-membranous cytoplasmic structures located in the perinuclear regions of germ cells. Thus, piRNA biogenesis is thought to occur in the nuage in germ cells. Immunofluorescence analyses of ovaries from piRNA factor mutants have revealed a localization hierarchy of piRNA factors in female nuage. However, whether this hierarchy is female-specific or can also be applied in male gonads remains undetermined. Here, we show by immunostaining of both ovaries and testes from piRNA factor mutants that the molecular hierarchy of piRNA factors shows gender-specificity, especially for Krimper (Krimp), a Tudor-domain-containing protein of unknown function(s): Krimp is dispensable for PIWI protein Aubergine (Aub) nuage localization in ovaries but Krimp and Aub require each other for their proper nuage localization in testes. This suggests that the functional requirement of Krimp in piRNA biogenesis may be different in male and female gonads.

Krimper Enforces an Antisense Bias on piRNA Pools by Binding AGO3 in the Drosophila Germline

“…In krimp mutant ovaries, AGO3 loses its ability to localize to the nuage (Nagao et al, 2011), while Aub remains accumulated in the nuage as in the control (Lim and Kai 2007;Nagao et al, 2011). These findings prompted us to examine if Krimp has a specific physical interaction with AGO3 in the ovary.…”

“…We found no significant association of Krimper with Dart5 and Valois, which are components of arginine-methylation complexes (methylosomes), (Anne and Mechler, 2005) (Figures S2F and S2G), suggesting that Krimp-AGO3 complexes only transiently interact with methylosomes. Because AGO3 lost its ability to localize to the nuage in krimp mutant ovaries (Malone et al, 2009;Nagao et al, 2011;Zhang et al, 2011), Krimp is likely to promote sDMA modification of AGO3 by localizing AGO3 in nuage where methylosome components are also accumulated (Anne and Mechler, 2005).…”

“…Furthermore, these findings suggest that primary piRNAs can be loaded onto AGO3, but that this loading is actively blocked in the presence of Aub. In aub mutant ovaries, the nuage localization of both AGO3 and Krimp is lost; instead, Krimp accumulates in large cytoplasmic foci termed Krimp bodies, where AGO3 is also accumulated (Lim and Kai, 2007;Nagao et al, 2011;Olivieri et al, 2012). Thus, the reduced loading of piRNAs onto AGO3 is probably caused by the sequestration of AGO3 to Krimp bodies.…”

“…In aub mutant ovaries, Krimp is no longer accumulated at the nuage but forms Krimp bodies (Lim and Kai, 2007;Nagao et al, 2011), suggesting that the nuage localization of Krimp is Aub dependent. However, Krimp does not directly interact with Aub, and Krimp in OSCs forms Krimp bodies when Aub is ectopically expressed.…”

The Drosophila fragile X mental retardation protein participates in the piRNA pathway

“…RESEARCH ARTICLE Journal of Cell Science (2015Science ( ) 128, 2070Science ( -2084Science ( doi:10.1242 in the male germline (Brennecke et al, 2007;Cook et al, 2004;Klattenhoff and Theurkauf, 2008;Nagao et al, 2011;Pane et al, 2007). Surprisingly, the conditional dFmr1 rescue and KD experiments demonstrate that dFmr1 controls the piRNA pathway both in the germline and in the somatic cells of the gonad, which raises questions as to the somatic contribution of other members of the piRNA pathway in the male gonad.…”