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Ribotrap: Targeted Purification of RNA-Specific RNPs from Cell Lysates Through Immunoaffinity Precipitation to Identify Regulatory Proteins and RNAs

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Post-Transcriptional Gene Regulation

Part of the book series: Methods In Molecular Biology™ ((MIMB,volume 419))

Summary

Many elegant methodologies have been devised to explore RNA-protein as well as RNA–RNA interactions. Although the characterization of messages targeted by a specific RNA-binding protein (RBP) has been accelerated by the application of microarray technologies, reliable methods to describe the endogenous assembly of ribonucleoproteins (RNPs) are needed. However, this approach requires the targeted purification of a select mRNA under conditions favorable for the copurification of associated factors including RNA and protein components of the RNP. This chapter describes previous methods used to characterize RNPs in the context of in vitro approaches and presents the Ribotrap methodology, an in vivo protocol for message-specific purification of a target RNP. The method was developed in a yeast model system, yet is amenable to other in vivo cell systems including mammalian cell culture.

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Acknowledgments

We thank the members of the Keene laboratory for intellectual support and stimulation, especially Kyle Mansfield and Matt Friedersdorf for critical review of the manuscript. We thank Kerry Bloom, University of North Carolina at Chapel Hill, for supplying yeast strains and plasmids. D.L.B. is supported by the Minority Opportunities in Research Division of the National Institute of General Medical Sciences (NIGMS) grant GM-000678.

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Authors and Affiliations

  1. University of North Carolina, Chapel Hill, USA

    Dale L. Beach

  2. Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA

    Dale L. Beach & Jack D. Keene

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  1. Dale L. Beach
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  2. Jack D. Keene
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Editors and Affiliations

  1. Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO, USA

    Jeffrey Wilusz

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© 2008 Humana Press, a part of Springer Science+Business Media, LLC

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Beach, D.L., Keene, J.D. (2008). Ribotrap: Targeted Purification of RNA-Specific RNPs from Cell Lysates Through Immunoaffinity Precipitation to Identify Regulatory Proteins and RNAs . In: Wilusz, J. (eds) Post-Transcriptional Gene Regulation. Methods In Molecular Biology™, vol 419. Humana Press. https://doi.org/10.1007/978-1-59745-033-1_5

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  • DOI: https://doi.org/10.1007/978-1-59745-033-1_5

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-783-9

  • Online ISBN: 978-1-59745-033-1

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