Euchromatic Transposon Insertions Trigger Production of Novel Pi- and Endo-siRNAs at the Target Sites in the Drosophila Germline

Abstract

The control of transposable element (TE) activity in germ cells provides genome integrity over generations. A distinct small RNA–mediated pathway utilizing Piwi-interacting RNAs (piRNAs) suppresses TE expression in gonads of metazoans. In the fly, primary piRNAs derive from so-called piRNA clusters, which are enriched in damaged repeated sequences. These piRNAs launch a cycle of TE and piRNA cluster transcript cleavages resulting in the amplification of piRNA and TE silencing. Using genome-wide comparison of TE insertions and ovarian small RNA libraries from two Drosophila strains, we found that individual TEs inserted into euchromatic loci form novel dual-stranded piRNA clusters. Formation of the piRNA-generating loci by active individual TEs provides a more potent silencing response to the TE expansion. Like all piRNA clusters, individual TEs are also capable of triggering the production of endogenous small interfering (endo-si) RNAs. Small RNA production by individual TEs spreads into the flanking genomic regions including coding cellular genes. We show that formation of TE-associated small RNA clusters can down-regulate expression of nearby genes in ovaries. Integration of TEs into the 3′ untranslated region of actively transcribed genes induces piRNA production towards the 3′-end of transcripts, causing the appearance of genic piRNA clusters, a phenomenon that has been reported in different organisms. These data suggest a significant role of TE-associated small RNAs in the evolution of regulatory networks in the germline.

Paramutation inDrosophilaRequires Both Nuclear and Cytoplasmic Actors of the piRNA Pathway and InducesCis-spreading of piRNA Production

“…The capacity to produce piRNAs invaded the whole I-derived transgene and reached in some cases flanking sequences. A similar spreading process into flanking sequences was also found for natural transposon insertions (Shpiz et al 2014). It is possible, however, that spreading processes inside or outside a transgene or a TE involve different mechanisms.…”

“…Figure 3C shows that, as early as generation 3, the three types of G 0 maternal inheritance resulted in strong production of small RNAs covering the entire BX2 P{lacW} transgene length. Thus, partially homologous paramutations are associated with rapid and stable spreading of piRNA production by nonhomologous sequences of the targets, similarly to what was found for target transgenes (Muerdter et al 2011;Olovnikov et al 2013;Han et al 2015;Mohn et al 2015) or transposable elements (Shpiz et al 2014).Further, the paramutagenicity of P-1152 BX2*, RS3 BX2*, and P-1152+RS3 BX2* females was tested. Figure 4 shows that maternal transmission of cytoplasm produced by these females, combined to paternal transmission of a BX2 naive locus, resulted in female progeny showing strong trans-silencing properties.…”

“…Further, these G 1 females were crossed with Dcr-2 +/+ males and increased ovarian P{lacW} siRNA levels were found in BX2* G 2 females having recovered a Dcr-2 +/+ genotype. Therefore, production of ovarian P{lacW} siRNAs by females having a functional siRNA pathway does not require homologous siRNA inheritance and can be induced by inheritance of solely homologous piRNAs.Paramutation of a locus only partially covered by maternally inherited piRNAs can be stable and is accompanied by cis-spreading of piRNA production capacityTargeting of transgenes or natural transposable elements by piRNAs can lead to spreading of piRNA production to sequences adjacent to the targeted elements (Muerdter et al 2011;Olovnikov et al 2013;Shpiz et al 2014). We tested if spreading could be observed for a silencer locus induced by paramutation.…”

“…Targeting of transgenes or natural transposable elements by piRNAs can lead to spreading of piRNA production to sequences adjacent to the targeted elements (Muerdter et al 2011;Olovnikov et al 2013;Shpiz et al 2014). We tested if spreading could be observed for a silencer locus induced by paramutation.…”

Reexamining the P-Element Invasion of Drosophila melanogaster Through the Lens of piRNA Silencing

“…Somewhat counterintuitively, H3K9me3 does not confer a transcriptionally-silenced state to piRNA clusters, but rather acts as a critical activator of piRNA biogenesis by recruiting proteins involved in piRNA cluster transcription and precursor processing (Rangan et al 2011;Le Thomas et al 2014a). The role of H3K9me3 in defining piRNA clusters may explain why euchromatic insertions of TE families, which are transcriptionally silenced by piRNAs through the deposition of H3K9me3, sometimes act like piRNA clusters; producing abundant piRNAs from both genomic strands (Shpiz et al 2014).…”