Dissection of a Natural RNA Silencing Process in the Drosophila melanogaster Germ Line

Abstract

To date, few natural cases of RNA-silencing-mediated regulation have been described. Here, we analyzed repression of testis-expressed Stellate genes by the homologous Suppressors of Stellate [Su(Ste)] repeats that produce sense and antisense short RNAs. The Stellate promoter is dispensable for suppression, but local disturbance of complementarity between the Stellate transcript and the Su(Ste) repeats impairs silencing. Using in situ RNA hybridization, we found temporal control of the expression and spatial distribution of sense and antisense Stellate and Su(Ste) transcripts in germinal cells. Antisense Su(Ste) transcripts accumulate in the nuclei of early spermatocytes before the appearance of sense transcripts. The sense and antisense transcripts are colocalized in the nuclei of mature spermatocytes, placing the initial step of silencing in the nucleus and suggesting formation of double-stranded RNA. Mutations in the aubergine and spindle-E genes, members of the Argonaute and RNA helicase gene families, respectively, impair silencing by eliminating the short Su(Ste) RNA, but have no effect on microRNA production. Thus, different small RNA-containing complexes operate in the male germ line.The silencing of genes by homologous double-stranded (ds) RNA (RNA interference [RNAi]) was discovered in artificial systems where dsRNA is introduced by injection or by expression of transgenic constructs (11,25,43). RNAi is widely used as a powerful technique for switching off specific genes and as a tool of whole-genome screening (12,22). The first case of natural dsRNA-mediated silencing was found in Drosophila melanogaster, where suppression of a euchromatic locus by homologous testis-expressed heterochromatic repeats has been shown to be necessary for male fertility (2,26,27). In testes of wild-type males, hyperexpression of the X-linked Stellate genes is prevented by the closely homologous, bidirectionally transcribed, Y-linked Supressor of Stellate [Su(Ste)] repeats (2, 26), and deletion of Su(Ste) leads to abnormalities of spermatogenesis (7, 35).Twenty-one-to 23-nucleotide (nt) short interfering (si) RNAs formed by processing of long dsRNA molecules by the RNase Dicer play a central role in dsRNA-mediated silencing (4,46,55). siRNA is assumed to act as a guide for the RNase complex (RISC) that degrades homologous mRNAs (9, 17, 33). microRNAs are involved in the control of expression of cellular genes (14,19,20,29). microRNAs are transcribed as precursors with a stem-loop structure that are processed by Dicer into single-stranded RNAs of a size similar to that of siRNA. Recent experiments suggest that siRNAs and microRNAs can be loaded into a common RISC (20,28,29,32). The Argonaute family of proteins represents the conserved core component of siRNA-and microRNA-containing complexes isolated from both D. melanogaster and mammals (18,21,29,32).The repression of Stellate in D. melanogaster by the closely homologous Su(Ste) repeats shares a number of traits with artificial RNAi. Bidirectional transcription of Su(Ste) re...

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Eukaryotic regulatory RNAs: an answer to the ‘genome complexity’ conundrum

“…These fragments then target the specific destruction of homologous mRNAs. Such sense-antisense RNA-induced gene silencing has been well documented in the case of the silencing of Drosophila stellate repeats (Aravin et al 2001(Aravin et al , 2004. Modulation of the hyperexpression of Drosophila stellate repeats in testis is essential for male fertility, and studies have shown that the Su(Ste) repeats produce both sense and antisense RNAs that form dsRNA in vivo (Aravin et al 2001).…”

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