Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes

Abstract

RNA interference (RNAi) is a mechanism by which double-stranded RNAs (dsRNAs) suppress specific transcripts in a sequence-dependent manner. dsRNAs are processed by Dicer to 21-24-nucleotide small interfering RNAs (siRNAs) and then incorporated into the argonaute (Ago) proteins. Gene regulation by endogenous siRNAs has been observed only in organisms possessing RNA-dependent RNA polymerase (RdRP). In mammals, where no RdRP activity has been found, biogenesis and function of endogenous siRNAs remain largely unknown. Here we show, using mouse oocytes, that endogenous siRNAs are derived from naturally occurring dsRNAs and have roles in the regulation of gene expression. By means of deep sequencing, we identify a large number of both approximately 25-27-nucleotide Piwi-interacting RNAs (piRNAs) and approximately 21-nucleotide siRNAs corresponding to messenger RNAs or retrotransposons in growing oocytes. piRNAs are bound to Mili and have a role in the regulation of retrotransposons. siRNAs are exclusively mapped to retrotransposons or other genomic regions that produce transcripts capable of forming dsRNA structures. Inverted repeat structures, bidirectional transcription and antisense transcripts from various loci are sources of the dsRNAs. Some precursor transcripts of siRNAs are derived from expressed pseudogenes, indicating that one role of pseudogenes is to adjust the level of the founding source mRNA through RNAi. Loss of Dicer or Ago2 results in decreased levels of siRNAs and increased levels of retrotransposon and protein-coding transcripts complementary to the siRNAs. Thus, the RNAi pathway regulates both protein-coding transcripts and retrotransposons in mouse oocytes. Our results reveal a role for endogenous siRNAs in mammalian oocytes and show that organisms lacking RdRP activity can produce functional endogenous siRNAs from naturally occurring dsRNAs.

New meaning in the message: Noncoding RNAs and their role in retinal development

“…This is shown for Prox1 and Hes5 in Figure 2. While it is unclear whether such duplexes actually form in vivo, naturally occurring sense-antisense pairs of this sort can inhibit translation (Werner and Berdal, 2005), and can also serve as substrates for the generation of endogenous siRNAs directed against the coding transcripts (Tam et al, 2008;Watanabe et al, 2008).…”

Mammalian microRNAs: experimental evaluation of novel and previously annotated genes

“…Those precursors bear little resemblance to the shorter, imperfectly base-paired hairpins of metazoan miRNA genes. In mice, similar precursors are found in the form of hairpin siRNA (hp-siRNA) precursors, but their expression appears to be limited to germline tissues and totipotent ES cells, which lack a robust interferon response to intracellular dsRNA (Babiarz et al 2008;Tam et al 2008;Watanabe et al 2008). However, we detected two miRNA:miRNA* duplexes deriving from the mmumir-3102 pre-miRNA hairpin, an apparent mirtron as evidenced by reads mapping to both boundaries of an For each sample, the fold enrichment of miRNA reads produced from the 59 arm over those produced from the 39 arm and vice versa was calculated.…”

Petits ARN non codants et spermatogenèse

“…Ces miARN matures s'associent à un 1 Figure 2B). Cette nouvelle classe de petits ARN non codants a été décrite dans les ovocytes [12], les cellules souches embryonnaires [13] et récemment dans les cellules germinales mâles [9]. Les données détaillant le rôle et l'importance de ces endo-siARN dans la régulation de la spermatogenèse sont toujours parcellaires, même si une étude récente a montré l'expression d'au moins 73 endo-siARN dans le testicule de souris, dont certains sont capables d'induire la dégradation d'ARNm cibles in vitro [9].…”

“…Les précurseurs des siARN endogènes sont des transcrits composés de longues structures en tête d'épingle ou d'ARN double brin dérivés de transcrits hybrides sens/anti-sens provenant du même locus (cis-endo-siARN) ou de deux locus différents (trans-endo-siARN). À partir de ces précurseurs sont produits les petits endo-siARN, un processus dépendant du clivage par DICER1 mais pas de DGCR8 [12]. C. Biogenèse des piARN.…”