Suffix-specific RNAi Leads to Silencing of F Element in Drosophila melanogaster

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Abstract
Separate conserved copies of suffix, a short interspersed Drosophila retroelement (SINE), and also divergent copies in the 3′ untranslated regions of the three genes, have already been described. Suffix has also been identified on the 3′ end of the Drosophila non-LTR F element, where it forms the last conserved domain of the reverse transcriptase (RT). In our current study, we show that the separate copies of suffix are far more actively transcribed than their counterparts on the F element. Transcripts from both strands of suffix are present in RNA preparations during all stages of Drosophila development, providing the potential for the formation of double-stranded RNA and the initiation of RNA interference (RNAi). Using in situ RNA hybridization analysis, we have detected the expression of both sense and antisense suffix transcripts in germinal cells. These sense and antisense transcripts are colocalized in the primary spermatocytes and in the cytoplasm of the nurse cells, suggesting that they form double-stranded RNA. We performed further analyses of suffix-specific small RNAs using northern blotting and SI nuclease protection assays. Among the total RNA preparations isolated from embryos, larvae, pupae and flies, suffix-specific small interfering RNAs (siRNAs) were detected only in pupae. In wild type ovaries, both the siRNAs and longer suffix-specific Piwi-interacting RNAs (piRNAs) were observed, whereas in ovaries of the Dicer-2 mutant, only piRNAs were detected. We further found by 3′ RACE that in pupae and ovaries, F element transcripts lacking the suffix sequence are also present. Our data provide direct evidence that suffix-specific RNAi leads to the silencing of the relative LINE (long interspersed element), F element, and suggests that SINE-specific RNA interference could potentially downregulate a set of genes possessing SINE stretches in their 5′ or 3′ non-coding regions. These data also suggest that double stranded RNAs possessing suffix are processed by both RNAi and an additional silencing mechanism.