Biogenesis pathways of piRNAs loaded onto AGO3 in the Drosophila testis

Abstract

PIWI-interacting RNAs (piRNAs) silence transposable elements in animal germ cells. In Drosophila ovaries, piRNAs are produced by two distinct pathways: the ''ping-pong'' amplification cycle that operates in germ cells and a ping-pongindependent pathway termed the primary pathway that mainly operates in somatic cells. AGO3, one of three PIWI proteins in flies, is involved in the ping-pong cycle in ovaries. We characterized AGO3-associated piRNAs in fly testes and found that like in ovaries, AGO3 functions in the ping-pong cycle with Aubergine (Aub) for piRNA production from transposon transcripts. In contrast, most AGO3-associated piRNAs corresponding to Suppressor of Stellate [Su(Ste)] genes are antisense-oriented and bound to Aub. In addition, the vast majority of AGO3-bound piRNAs derived from the AT-chX locus on chromosome X are antisense-oriented and are also found among Aub-associated piRNAs. The presence of very few sense Su(Ste) and AT-chX piRNAs suggests that biogenesis of both Su(Ste) and AT-chX piRNAs by a ping-pong mechanism only is highly unlikely. Nevertheless, the mutual interdependence of AGO3 and Aub for the accumulation of these piRNAs shows that their production relies on both AGO3 and Aub. Analysis of piRNA pathway mutants revealed that although the requirements for piRNA factors for Su(Ste)-and AT-chX-piRNA levels mostly overlap and resemble those for the ping-pong mechanism in the ovaries, Armitage (armi ) is not required for the accumulation of AT-chX-1 piRNA. These findings suggest that the impacts of armi mutants on the operation of the piRNA pathway are variable in germ cells of fly testes.

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

“…Silencing of repetitive transposon targets in the germline of both testes and ovaries utilizes a mechanism known as "ping-pong" piRNA production because it requires reciprocal cleavage of sense and antisense RNAs by piRNA-guided AGO3 and Aub proteins, respectively Gunawardane et al 2007;Nagao et al 2010). 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).…”

“…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). Interestingly, Rsp s piRNAs have mostly been detected in association with AGO3 in wild-type testes.…”

“…In the somatic cells of the ovary Zuc functions in production of Piwi-associated primary piRNAs and may serve to promote localization of the piRNA processing machinery to mitochondria Malone et al 2009;Saito et al 2009;Haase et al 2010;Watanabe et al 2011;Nishimasu et al 2012). The role of Zuc in the testis has not been extensively studied; however, loss of zuc has been shown to have little effect on the abundance of ATchX-1 and Su(ste)-4, the two most common piRNAs in the Drosophila testes (Nagao et al 2010).…”

“…Interestingly, Squ is a component of a testis-specific, electron-dense, perinuclear structure known as the piRNA Nuage Giant Body (piNG-body), thought to be a major site of piRNA processing and/or targeting in spermatocytes (Kibanov et al 2011). Squ mutant males exhibit a small reduction in levels of At-chX-1 and Su(ste)-4 piRNAs (Nagao et al 2010). In squ mutant females overall piRNA levels remain largely the same, with a slight decrease detected in some studies Malone et al 2009;Haase et al 2010).…”

“…In support of this hypothesis, Rsp sequence has been found as piRNA associated with both Aub and AGO3 in Drosophila testes (Nagao et al 2010). Further, two other well-studied meiotic drive systems, the Stellate system in D. melanogaster and the Winters system in D. simulans, are thought to involve small RNA-based silencing mechanisms (Aravin et al 2001(Aravin et al , 2004 Tao et al 2007a,b).…”