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Identification of mRNA-Interacting Factors by MS2-TRAP (MS2-Tagged RNA Affinity Purification)

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RNA-Protein Complexes and Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1421))

Abstract

Posttranscriptional gene expression is governed by the interaction of mRNAs with vast families of RNA-binding proteins (RBPs) and noncoding (nc)RNAs. RBPs and ncRNAs jointly influence all aspects of posttranscriptional metabolism, including pre-mRNA splicing and maturation, mRNA transport, editing, stability, and translation. Given the impact of mRNA-interacting molecules on gene expression, there is great interest in identifying mRNA-binding factors comprehensively. Here, we provide a detailed protocol to tag mRNAs with MS2 hairpins and then affinity-purify trans-binding factors (RBPs, ncRNAs) associated with the MS2-tagged mRNA. This method, termed MS2-TRAP, permits the systematic characterization of ribonucleoprotein (RNP) complexes formed on a given mRNA of interest. We describe how to prepare the mRNA-MS2 expression vector, purify the MS2-tagged RNP complexes, and detect bound RNAs and RBPs, as well as variations of this methodology to address related questions of RNP biology.

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Acknowledgments

J.H.Y. and M.G. were supported by the National Institute on Aging Intramural Research Program, National Institutes of Health and start-up fund from Medical University of South Carolina.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC, 29425, USA

    Je-Hyun Yoon

  2. Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Laboratory of Genetics and Genomics, National Institute on Aging-Intramural Research Program, National Institutes of Health, 173 Ashley Avenue, Charleston, SC, 29425, USA

    Myriam Gorospe

Authors
  1. Je-Hyun Yoon
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  2. Myriam Gorospe
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Corresponding author

Correspondence to Je-Hyun Yoon .

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Editors and Affiliations

  1. Beckman res.Ins.Dept.of Mol. & Cell Bio., Irell&Manella.Grad.Schl.Biolog.Sci., Duarte, California, USA

    Ren-Jang Lin

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Yoon, JH., Gorospe, M. (2016). Identification of mRNA-Interacting Factors by MS2-TRAP (MS2-Tagged RNA Affinity Purification). In: Lin, RJ. (eds) RNA-Protein Complexes and Interactions. Methods in Molecular Biology, vol 1421. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3591-8_2

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  • DOI: https://doi.org/10.1007/978-1-4939-3591-8_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3589-5

  • Online ISBN: 978-1-4939-3591-8

  • eBook Packages: Springer Protocols

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