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Purification of Transcript-Specific mRNP Complexes Formed In Vivo from Saccharomyces cerevisiae

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mRNA Processing

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

Abstract

RNA binding proteins play critical roles in shaping the complex life cycle of cellular transcripts. For most RNAs, the association with a distinct complement of proteins serves to orchestrate its unique pattern of maturation, localization, translation, and stability. A key aspect to understanding how transcripts are differentially regulated lies, therefore, in the ability to identify the particular repertoire of protein binding partners associated with an individual transcript. We describe here an optimized experimental procedure for purifying a single mRNA population from yeast cells for the characterization of transcript-specific mRNA-protein complexes (mRNPs) as they exist in vivo. Chemical cross-linking is used to trap native mRNPs and facilitate the co-purification of protein complexes associated with an individual transcript population that is captured under stringent conditions from cell lysates through hybridization to complementary DNA oligonucleotides. The resulting mRNP is highly enriched and largely devoid of non-target transcripts, and can be used for a number of downstream analyses including protein identification by mass spectrometry.

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Acknowledgments

We thank Tim Nilsen and members of his lab for providing helpful suggestions during the development of this method. Jeff Coller and Coller lab member, Najwa Alhusaini, provided helpful comments and critical reading of this manuscript. Mass spectrometry and identification of protein components within our transcript-specific mRNPs was achieved in collaboration with Amber Mosley and Whitney Smith-Kinnaman in the Department of Biochemistry and Molecular Biology at the Indiana University School of Medicine. This work was supported by funding by the National Institute of General Medical Sciences (GM095621 to K.E.B.; T32 GM008056 to J.E.S.) and the National Science Foundation (NSF1253788 to K.E.B.).

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Authors and Affiliations

  1. Center for RNA Science and Therapeutics, Case Western Reserve University School of Medicine, Cleveland, OH, 44106, USA

    Jenna E. Smith & Kristian E. Baker

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  1. Jenna E. Smith
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  2. Kristian E. Baker
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Correspondence to Kristian E. Baker .

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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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Smith, J.E., Baker, K.E. (2017). Purification of Transcript-Specific mRNP Complexes Formed In Vivo from Saccharomyces cerevisiae . 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_15

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  • DOI: https://doi.org/10.1007/978-1-4939-7204-3_15

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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

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