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

Abstract

Piwi-interacting RNAs (piRNAs) suppress transposon activity in animal germ cells. In the Drosophila ovary, primary Aubergine (Aub)-bound antisense piRNAs initiate the ping-pong cycle to produce secondary AGO3-bound sense piRNAs. This increases the number of secondary Aub-bound antisense piRNAs that can act to destroy transposon mRNAs. Here we show that Krimper (Krimp), a Tudor-domain protein, directly interacts with piRNA-free AGO3 to promote symmetrical dimethylarginine (sDMA) modification, ensuring sense piRNA-loading onto sDMA-modified AGO3. In aub mutant ovaries, AGO3 associates with ping-pong signature piRNAs, suggesting AGO3's compatibility with primary piRNA loading. Krimp sequesters ectopically expressed AGO3 within Krimp bodies in cultured ovarian somatic cells (OSCs), in which only the primary piRNA pathway operates. Upon krimp-RNAi in OSCs, AGO3 loads with piRNAs, further showing the capacity of AGO3 for primary piRNA loading. We propose that Krimp enforces an antisense bias on piRNA pools by binding AGO3 and blocking its access to primary piRNAs.

Loss of l(3)mbt leads to acquisition of the ping-pong cycle in Drosophila ovarian somatic cells

“…S1B). Western blotting detected AGO3, Vasa, and Aub in the RNAi-treated cells, while the levels of Piwi, Yb, Armi, and Krimper (Krimp) (Saito et al 2009;Iwasaki et al 2015;Sato et al 2015) remained unchanged (Supplemental Fig. S1C).…”

“…We previously showed that both Myc-Aub and Myc-AGO3 exogenously expressed in OSCs by transfection are loaded with primary piRNAs, including flam-piRNAs and tj-piRNAs, which are normally loaded onto endogenous Piwi (Olivieri et al 2012;Sato et al 2015). This emphasized the compatibility of AGO3 with primary piRNA loading.…”

“…However, in Δmbt-OSCs, AGO3 avoided binding primary piRNAs; this was especially apparent when tjpiRNAs were compared (Supplemental Fig. S3C; Sato et al 2015). In OSCs, Krimp sequesters unloaded AGO3 to Krimp bodies and blocks AGO3-primary piRNA loading (Olivieri et al 2012;Sato et al 2015).…”

The piRNA pathway is developmentally regulated during spermatogenesis in Drosophila

“…Nuage (which means "cloud" in French) is a conserved perinuclear structure present at the cytoplasmic face of the nuclear envelope in germline cells (Eddy 1975). Interestingly, in Drosophila, many of the nuage proteins have been involved in the production of piRNAs, such as DEAD-box RNA helicases like Vasa (Liang et al 1994), PIWI family proteins Aub and Ago3 (Harris and Macdonald 2001;Brennecke et al 2007;Gunawardane et al 2007), and many Tudor domain proteins such as Krimper (Krimp) (Lim and Kai 2007;Sato et al 2015) and Qin/Kumo Anand and Kai 2012). Thus, nuage was proposed as the potential site for a ping-pong amplification loop in the germline.…”

Novel mutants of theauberginegene

“…22 At our growing conditions aub QC42 is not completely vital, only few escapers survive; as a consequence we and other groups routinely used the aub HN2 /aub QC42 transheterozygotes, in order to analyze aub "loss of function." 3,[23][24][25] For the above reasons, we decided to produce novel and null aub alleles by mobilizing the P element transposon present in the P{lacW} aub sting allele, also known as aub sting . P-element mutagenesis is a convenient method to obtain knock-out mutants.…”