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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) proteins1,2,3,4. Gene regulation by endogenous siRNAs has been observed only in organisms possessing RNA-dependent RNA polymerase (RdRP)5,6,7,8,9,10. 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 25–27-nucleotide Piwi-interacting RNAs (piRNAs) and 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.

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Figure 1: Small RNA profile and expression of small RNA pathway components in mouse oocytes.
Figure 2: Structure of the hp-siRNA cluster at the Au76 locus.
Figure 3: Structure of the cis-nat-siRNA cluster at the Pdzd11/Kif4 locus.
Figure 4: Structure of the trans-nat-siRNA cluster pair at the loci of Ppp4r1 and its processed pseudogene.

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

The GenBank accession numbers of the small RNAs mapped to the genome in clusters and piRNAs co-immunoprecipitated with Mili are AB334800–AB349184 and AB349185–AB353040, respectively.

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Acknowledgements

We thank A. Tarakhovsky and D. O’Carroll for mice with the Dicer and Ago2 conditional alleles; G. J. Hannon and A. Girard for the anti-AGO2 antibody; T. Sado, K. Hata and H. Furuumi for scientific and technical advice; Y. Kurihara, A. Takeda and K. Ichiyanagi for comments on the manuscript; N. Minami and Y. Hoki for expertise in mouse oocytes; and K. Takada and M. Kiyooka for technical assistance. We thank RIKEN for the Super Combined Cluster (RSCC) computational resources. We also thank members of the Sasaki laboratory for discussion and encouragement. T.W. is a research fellow of the Japan Society for the Promotion of Science. This work was supported in part by Grants-in-Aid for Scientific Research on Priority Area from the Ministry of Education, Culture, Sports, Science, and Technology of Japan to H.S.

Author Contributions T.W. performed experiments and interpreted results; Y.T. generated computational programs and analysed data with T.W.; A.T., Y.S. and Y.K. were involved in small RNA sequencing; Y.O., H.C. and T.K. were involved in oocyte collection; M.K. and M.A.S. prepared the samples from conditional Dicer and Ago2 knockout mice; S.K.-M. and T.N. provided samples from Mili knockout mouse; H.S. and T.W. designed the study and wrote the manuscript.

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Correspondence to Toshiaki Watanabe or Hiroyuki Sasaki.

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Watanabe, T., Totoki, Y., Toyoda, A. et al. Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453, 539–543 (2008). https://doi.org/10.1038/nature06908

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