The endonuclease activity of Mili fuels piRNA amplification that silences LINE1 elements

Abstract

Piwi proteins and Piwi-interacting RNAs (piRNAs) have conserved functions in transposon silencing. The murine Piwi proteins Mili and Miwi2 (also called Piwil2 and Piwil4, respectively) direct epigenetic LINE1 and intracisternal A particle transposon silencing during genome reprogramming in the embryonic male germ line. Piwi proteins are proposed to be piRNA-guided endonucleases that initiate secondary piRNA biogenesis; however, the actual contribution of their endonuclease activities to piRNA biogenesis and transposon silencing remain unknown. To investigate the role of Piwi-catalysed endonucleolytic activity, we engineered point mutations in mice that substitute the second aspartic acid to an alanine in the DDH catalytic triad of Mili and Miwi2, generating the Mili(DAH) and Miwi2(DAH) alleles, respectively. Analysis of Mili-bound piRNAs from homozygous Mili(DAH) fetal gonadocytes revealed a failure of transposon piRNA amplification, resulting in the marked reduction of piRNA bound within Miwi2 ribonuclear particles. We find that Mili-mediated piRNA amplification is selectively required for LINE1, but not intracisternal A particle, silencing. The defective piRNA pathway in Mili(DAH) mice results in spermatogenic failure and sterility. Surprisingly, homozygous Miwi2(DAH) mice are fertile, transposon silencing is established normally and no defects in secondary piRNA biogenesis are observed. In addition, the hallmarks of piRNA amplification are observed in Miwi2-deficient gonadocytes. We conclude that cycles of intra-Mili secondary piRNA biogenesis fuel piRNA amplification that is absolutely required for LINE1 silencing.

Roles for small noncoding RNAs in silencing of retrotransposons in the mammalian brain

“…Furthermore, consistent with the ping-pong feature, LINE1 and SINE secondary piRNA fractions had bias for small RNAs in antisense orientation (antisense/sense = 1.9 for LINE1, 1.7 For SINE, and 1.0 for LTR), in contrast to the endo-siRNA fraction (antisense/sense = 0.9 for LINE1, 0.6 For SINE, and 0.8 for LTR) (1). These results were consistent with plausible Milidirected piRNA amplification in the brain similar to what has been described for the fetal germline (1,34).…”

“…2A and Dataset S3). Consistent with the "ping-pong" feature associated with biogenesis of LINE1-derived germline piRNAs, a small but distinct peak ∼28 nt was visible for neuronal LINE1 clones that lacked 5′ uridine but had adenine at the 10th position (No-1U, 10A, or secondary piRNA fraction), again with maximum abundance of L1Md_F2 (1,34) (Fig. 2B and Dataset S3).…”

Targeted gene silencing in mouse germ cells by insertion of a homologous DNA into a piRNA generating locus

“…In Drosophila, zebrafish, and Xenopus, a majority of piRNAs has sequences corresponding to transposable elements including retrotransposons (Vagin et al 2006), and mutations in genes encoding the piRNA pathway components result in their derepression. The PIWI-piRNA complexes recognize their target RNAs based on sequence complementarity and cause RNA degradation (post-transcriptional gene silencing or PTGS) (Lim et al 2009;De Fazio et al 2011;Reuter et al 2011). In fetal mouse testes, the complexes also repress retrotransposons transcriptionally by introducing DNA methylation (transcriptional gene silencing or TGS) (Kuramochi-Miyagawa et al 2008).…”

“…It is generally believed that the PIWI-piRNA complexes regulate gene expression by cleaving target RNAs (PTGS) (Brennecke et al 2007;Gunawardane et al 2007;De Fazio et al 2011;Reuter et al 2011). In round spermatids, PIWIL2 and PIWIL1 proteins are localized to chromatoid bodies implicated in RNA processing and PTGS (Chuma et al 2009).…”

Les piARN forgent un système immunitaire pour le génome

“…Pour cela, il est néces-saire de générer un piARN primaire ou secondaire capable de s'apparier à la cible (piARN antisens), mais aussi de recruter une protéine PIWI catalytiquement active. Ce dernier point a été examiné en mutant la triade catalytique DDH des protéines PIWI et en mesurant les effets sur les populations de piARN présentant les signatures précédemment énoncées [18,19]. déposés dans l'embryon et peuvent initier un cycle d'amplification qui permet aux lignées germinales de la génération suivante de se défendre contre les éléments I [27].…”