Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways 1 . Here we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from proteincoding genes to antisense transcripts from homologous pseudogenes. An inverted repeat pseudogene can also generate abundant small RNAs directly. A second class of endo-siRNAs may enforce repression of mobile genetic elements, acting together with Piwi-interacting RNAs. Loss of Dicer, a protein integral to small RNA production, increases expression of endo-siRNA targets, demonstrating their regulatory activity. Our findings indicate a function for pseudogenes in regulating gene expression by means of the RNA interference pathway and may, in part, explain the evolutionary pressure to conserve argonaute-mediated catalysis in mammals.Small-RNA-directed gene silencing pathways have been adapted to accept numerous inputs and to act on many types of downstream targets. In few places is this more apparent than in animal germ lines where two classes of small RNAs-microRNAs (miRNAs) and Piwiinteracting RNAs (piRNAs)-with distinct biogenesis mechanisms and biological functions have been reported. Although miRNAs, as a group, are ubiquitously expressed, piRNAs have thus far been found only in germ cells and in a few gonadal somatic cells types 2 . piRNAs repress the activity of mobile genetic elements, forming a small RNA-based, innate immune system with both genetically encoded and adaptive components2 -9.In mice, a homozygous mutation in any single Piwi family member causes male sterility accompanied by gonadal hypotrophy 5 , 10 , 11. In Mili and Miwi2 mutants, meiosis is not completed and germ cells are progressively lost5. This correlates with an activation of transposons, particularly the non-long terminal repeat (LTR) retrotransposon, L1 (refs 5 , 12).
Dicer is an RNase III-family nuclease that initiates RNA interference (RNAi) and related phenomena by generation of the small RNAs that determine the specificity of these gene silencing pathways. We have previously shown that Dicer is essential for mammalian development, with Dicer-deficient mice dying at embryonic day 7.5 with a lack of detectable multipotent stem cells. To permit a more detailed investigation of the biological roles of Dicer, we have generated embryonic stem cell lines in which their single Dicer gene can be conditionally inactivated. As expected, Dicer loss compromises maturation of microRNAs and leads to a defect in gene silencing triggered by long dsRNAs. However, the absence of Dicer does not affect the ability of small interfering RNAs to repress gene expression. Of interest, Dicer loss does compromise the proliferation of ES cells, possibly rationalizing the phenotype previously observed in Dicer-null animals. Dicer loss also affects the abundance of transcripts from mammalian centromeres but does so without a pronounced affect on histone modification status at pericentric repeats or methylation of centromeric DNA. These studies provide a conditional model of RNAi deficiency in mammals that will permit the dissection of the biological roles of the RNAi machinery in cultured mammalian cells.centromeres ͉ RNA interference
Supplementary data are available at Bioinformatics online.
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