Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes (original) (raw)

• Letter
• Published: 10 April 2008
• Yasushi Totoki3 nAff10,
• Atsushi Toyoda4,
• Masahiro Kaneda5,6,
• Satomi Kuramochi-Miyagawa7,
• Yayoi Obata8,
• Hatsune Chiba1,2,
• Yuji Kohara2,9,
• Tomohiro Kono8,
• Toru Nakano7,
• M. Azim Surani8,
• Yoshiyuki Sakaki3,4 &
• …
• Hiroyuki Sasaki1,2

Nature volume 453, pages 539–543 (2008)Cite this article

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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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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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Author notes

1. Yasushi Totoki
   Present address: Present address: MetaSystems Research Team, RIKEN Advanced Science Institute, Yokohama 230-0045, Japan.,

Authors and Affiliations

1. Division of Human Genetics, Department of Integrated Genetics, National Institute of Genetics, Research Organization of Information and Systems, Mishima 411-8540, Japan,
   Toshiaki Watanabe, Hatsune Chiba & Hiroyuki Sasaki
2. Department of Genetics, School of Life Science, The Graduate University for Advanced Studies (SOKENDAI), Mishima 411-8540, Japan
   Toshiaki Watanabe, Hatsune Chiba, Yuji Kohara & Hiroyuki Sasaki
3. Genome Annotation and Comparative Analysis Team, Computational and Experimental Systems Biology Group, and,,
   Yasushi Totoki & Yoshiyuki Sakaki
4. Sequence Technology Team, RIKEN Genomic Sciences Center, Yokohama 230-0045, Japan ,
   Atsushi Toyoda & Yoshiyuki Sakaki
5. Wellcome Trust/Cancer Research UK Gurdon Institute of Cancer and Developmental Biology, University of Cambridge, Cambridge CB2 1QN, UK
   Masahiro Kaneda
6. Reproductive Biology and Technology Research Team, National Institute of Livestock and Grassland Science, National Agriculture and Food Research Organization, Tsukuba 305-0901, Japan ,
   Masahiro Kaneda
7. Department of Pathology, Graduate School of Medicine and Frontier Biosciences, Osaka University, Osaka 565-0871, Japan
   Satomi Kuramochi-Miyagawa & Toru Nakano
8. Department of BioScience, Tokyo University of Agriculture, Tokyo 156-8502, Japan
   Yayoi Obata, Tomohiro Kono & M. Azim Surani
9. Genome Biology Laboratory, Center for Genetic Resource Information, National Institute of Genetics, Research Organization of Information and Systems, Mishima 411-8540, Japan ,
   Yuji Kohara

Authors

1. Toshiaki Watanabe
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2. Yasushi Totoki
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3. Atsushi Toyoda
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4. Masahiro Kaneda
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5. Satomi Kuramochi-Miyagawa
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6. Yayoi Obata
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7. Hatsune Chiba
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9. Tomohiro Kono
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10. Toru Nakano
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11. M. Azim Surani
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Correspondence toToshiaki 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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• Received: 01 November 2007
• Accepted: 10 March 2008
• Published: 10 April 2008
• Issue Date: 22 May 2008
• DOI: https://doi.org/10.1038/nature06908

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Editorial Summary

Pseudogenes: Not without influence

Over evolutionary time, many genes undergo duplication and one copy accumulates mutations that render it non-functional. These 'pseudogenes' are generally thought to be rather uninteresting, dead-end pieces of the genome. Yet there now appears to be more to it than that. Two groups report in this issue on pseudogenes that can in fact influence gene expression. The mechanism involves pairing of RNA antisense transcripts from pseudogenes with the mRNAs of protein-coding genes, forming a duplex RNA that is processed into endogenous siRNAs.