The endogenous siRNA pathway is involved in heterochromatin formation in Drosophila

Abstract

A new class of small RNAs (endo-siRNAs) produced from endogenous double-stranded RNA (dsRNA) precursors was recently shown to mediate transposable element (TE) silencing in the Drosophila soma. These endo-siRNAs might play a role in heterochromatin formation, as has been shown in S. pombe for siRNAs derived from repetitive sequences in chromosome pericentromeres. To address this possibility, we used the viral suppressors of RNA silencing B2 and P19. These proteins normally counteract the RNAi host defense by blocking the biogenesis or activity of virus-derived siRNAs. We hypothesized that both proteins would similarly block endo-siRNA processing or function, thereby revealing the contribution of endo-siRNA to heterochromatin formation. Accordingly, P19 as well as a nuclear form of P19 expressed in Drosophila somatic cells were found to sequester TE-derived siRNAs whereas B2 predominantly bound their longer precursors. Strikingly, B2 or the nuclear form of P19, but not P19, suppressed silencing of heterochromatin gene markers in adult flies, and altered histone H3-K9 methylation as well as chromosomal distribution of histone methyl transferase Su(var)3-9 and Heterochromatin Protein 1 in larvae. Similar effects were observed in dcr2, r2d2, and ago2 mutants. Our findings provide evidence that a nuclear pool of TE-derived endo-siRNAs is involved in heterochromatin formation in somatic tissues in Drosophila.RNAi ͉ nucleus ͉ viruses R ecent deep sequencing efforts have provided critical information on Drosophila small RNA repertoires in various tissues and during distinct developmental stages (1-7). Four classes of small RNAs mediate posttranscriptional gene silencing in Drosophila: i) Ϸ22-nt miRNAs are processed from stem-loop precursors by Dicer-1 and repress mRNA expression; ii) Ϸ25-nt piRNAs are produced from transposable element (TE) transcripts in gonads where they silence TEs through a feedback regulatory mechanism involving the PIWI subfamily of Argonautes (2, 3, 8-11); iii) 21-nt siRNAs are processed from long dsRNAs by Dicer-2 and trigger RNAi, for instance in response to viral infection (12-14); and iv) recently discovered 21-nt endo-siRNAs are processed from endogenous dsRNA precursors by Dicer-2 and silence TEs, and possibly endogenous mRNA in somatic tissues (1,(5)(6)(7)15).In S. pombe, siRNAs produced from repetitive sequences in chromosome pericentromeres direct heterochromatin formation and transcriptional gene silencing. As in S. pombe (16), Drosophila heterochromatin is prominent in pericentromeric regions, mostly comprised of short satellite repeats and TEs, and is associated with histone H3 methylation on lysine 9 (H3K9) by the histone methylase Su(var)3-9 (Clr4 in S pombe). This allows recruitment of the Heterochromatin Protein HP1 (SWI6 in S. pombe) to maintain and spread heterochromatin to nearby genes (17). Despite these analogies, the evidence supporting a role of small RNAs in heterochromatin formation and transcriptional gene silencing in Drosophila remain indirect (18,19). Mutants for the ...

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Male germ cells express abundant endogenous siRNAs

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