suffix-specific rnai leads to silencing of f element in drosophila melanogastersuffix-specific rnai导致沉默f元素黑腹果蝇.pdf

Suffix-specific RNAi Leads to Silencing of F Element in Drosophila melanogaster Nickolai A. Tchurikov*, Olga V. Kretova Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia Separate conserved copies of suffix, a short interspersed Drosophila retroelement (SINE), and also divergent copies in the 39 untranslated regions of the three genes, have already been described. Suffix has also been identified on the 39 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 39 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 l