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Generation of Endo-siRNAs in Xenopus laevis Oocytes

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Animal Endo-SiRNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1173))

Abstract

Endogenous siRNAs (endo-siRNAs) are well documented and characterized in C. elegans and Drosophila. Endo-siRNAs can also be found in vertebrates; however, their biology is much less clear. They are thought to be produced by Dicer and to contribute to transposon silencing. Because of their generally low abundance and their similarity with miRNAs and products of physiological RNA turn-over, endo-siRNAs are difficult to investigate. Here, we report a system, oocytes from Xenopus laevis, that allows for the generation and analysis of endo-siRNAs from double-stranded RNA precursors.

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References

  1. Okamura K, Lai EC (2008) Endogenous small interfering RNAs in animals. Nat Rev Mol Cell Biol 9:673–678

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  2. Chapman EJ, Carrington JC (2007) Specialization and evolution of endogenous small RNA pathways. Nat Rev Genet 8:884–896

    Article  CAS  PubMed  Google Scholar 

  3. Wang Q, Carmichael GG (2004) Effects of length and location on the cellular response to double-stranded RNA. Microbiol Mol Biol Rev 68:432–452

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Tam OH, Aravin AA, Stein P, Girard A, Murchison EP, Cheloufi S, Hodges E, Anger M, Sachidanandam R, Schultz RM, Hannon GJ (2008) Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes. Nature 453:534–538

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  5. Carlile M, Swan D, Jackson K, Preston-Fayers K, Ballester B, Flicek P, Werner A (2009) Strand selective generation of endo-siRNAs from the Na/phosphate transporter gene Slc34a1 in murine tissues. Nucleic Acids Res 37:2274–2282

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Watanabe T, Totoki Y, Toyoda A, Kaneda M, Kuramochi-Miyagawa S, Obata Y, Chiba H, Kohara Y, Kono T, Nakano T, Surani MA, Sakaki Y, Sasaki H (2008) Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes. Nature 453:539–543

    Article  CAS  PubMed  Google Scholar 

  7. Soumillon M, Necsulea A, Weier M, Brawand D, Zhang X, Gu H, Barthes P, Kokkinaki M, Nef S, Gnirke A, Dym M, de Massy B, Mikkelsen TS, Kaessmann H (2013) Cellular source and mechanisms of high transcriptome complexity in the Mammalian testis. Cell Rep 3:2179–2190

    Article  CAS  PubMed  Google Scholar 

  8. Carlile M, Nalbant P, Preston-Fayers K, McHaffie GS, Werner A (2008) Processing of naturally occurring sense/antisense transcripts of the vertebrate Slc34a gene into short RNAs. Physiol Genomics 34:95–100

    Article  CAS  PubMed  Google Scholar 

  9. Saccomanno L, Bass BL (1994) The cytoplasm of Xenopus oocytes contains a factor that protects double-stranded RNA from adenosine-to-inosine modification. Mol Cell Biol 14:5425–5432

    CAS  PubMed Central  PubMed  Google Scholar 

  10. Markovich D (2008) Expression cloning and radiotracer uptakes in Xenopus laevis oocytes. Nat Protoc 3:1975–1980

    Article  CAS  PubMed  Google Scholar 

  11. Tammaro P, Shimomura K, Proks P (2008) Xenopus oocytes as a heterologous expression system for studying ion channels with the patch-clamp technique. Methods Mol Biol 491:127–139

    Article  CAS  PubMed  Google Scholar 

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Acknowledgment

This work has been supported by the Dunhill Medical Trust and Newcastle University.

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Correspondence to Andreas Werner .

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© 2014 Springer Science+Business Media New York

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Alnumeir, S., Werner, A. (2014). Generation of Endo-siRNAs in Xenopus laevis Oocytes. In: Werner, A. (eds) Animal Endo-SiRNAs. Methods in Molecular Biology, vol 1173. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0931-5_3

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  • DOI: https://doi.org/10.1007/978-1-4939-0931-5_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-0930-8

  • Online ISBN: 978-1-4939-0931-5

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