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Purification of mRNA Processing Complexes Using an RNA Affinity Approach

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mRNA Processing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1648))

Abstract

Multiple mRNA processing steps, including splicing and 3′ processing, take place in macromolecular complexes that contain many proteins and sometimes RNA molecules. A key challenge in the mRNA processing field has been to define the structure-function relationship of these sophisticated molecular machines. A prerequisite for addressing this challenge is to develop tools for purifying mRNA processing complexes in their native and intact forms that are suitable for functional and structural studies. Among many methods that have been developed, RNA affinity-based methods are most widely applied. In these methods, RNA molecules that are substrates to mRNA processing machineries are fused with an affinity tag, incubated with cellular extracts/lysates to allow for the assembly of mRNA processing complexes, and finally the assembled complexes are purified using RNA affinity tag. In this chapter, we will overview RNA affinity-based purification methods and describe in detail one such method, MS2-tagging, and its application in the purification of mRNA 3′ processing complexes. Although these methods were originally developed for purifying mRNA processing complexes, they should be applicable to purification of other RNA-protein complexes as well.

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Acknowledgment

This study was supported by grants from the National Institutes of Health (GM090056 and CA177651) and the American Cancer Society (RSG-12-186).

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Authors and Affiliations

  1. Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, Irvine, CA, 92697, USA

    Xiuye Wang & Yongsheng Shi

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  1. Xiuye Wang
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  2. Yongsheng Shi
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Correspondence to Yongsheng Shi .

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Editors and Affiliations

  1. Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, California, USA

    Yongsheng Shi

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Wang, X., Shi, Y. (2017). Purification of mRNA Processing Complexes Using an RNA Affinity Approach. In: Shi, Y. (eds) mRNA Processing. Methods in Molecular Biology, vol 1648. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7204-3_5

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  • DOI: https://doi.org/10.1007/978-1-4939-7204-3_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7203-6

  • Online ISBN: 978-1-4939-7204-3

  • eBook Packages: Springer Protocols

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