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Optimizing In Vitro Pre-mRNA 3′ Cleavage Efficiency: Reconstitution from Anion-Exchange Separated HeLa Cleavage Factors and from Adherent HeLa Cell Nuclear Extract

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Eukaryotic Transcriptional and Post-Transcriptional Gene Expression Regulation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1507))

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Abstract

Eukaryotic RNA processing steps during mRNA maturation present the cell with opportunities for gene expression regulation. One such step is the pre-mRNA 3′ cleavage reaction, which defines the downstream end of the 3′ untranslated region and, in nearly all mRNA, prepares the message for addition of the poly(A) tail. The in vitro reconstitution of 3′ cleavage provides an experimental means to investigate the roles of the various multi-subunit cleavage factors. Anion-exchange chromatography is the simplest procedure for separating the core mammalian cleavage factors. Here we describe a method for optimizing the in vitro reconstitution of 3′ cleavage activity from the DEAE-sepharose separated HeLa cleavage factors and show how to ensure, or avoid, dependence on creatine phosphate. Important reaction components needed for optimal processing are discussed. We also provide an optimized procedure for preparing small-scale HeLa nuclear extracts from adherent cells for use in 3′ cleavage in vitro.

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Acknowledgments

This work was supported by grant 5SC1GM083754 to K.R. from the National Institutes of Health. Additional infrastructural support at the City College of New York was provided by the NIH National Center for Research Resources (2G12RR03060-26A1) and the National Institute on Minority Health and Health Disparities (8G12MD007603-27).

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, The City College of New York, 160 Covent Avenue, New York, NY, 10031, USA

    Mihwa Na & Kevin Ryan

  2. Biochemistry Ph.D. Program, The City University of New York Graduate Center, New York, NY, 10016, USA

    Mihwa Na & Kevin Ryan

  3. Department of Immunology and Microbial Sciences, The Scripps Research Institute, Jupiter, FL, USA

    Susana T. Valente

  4. Program in Chemistry, The Graduate Center of the City, University of New York, New York, NY, 10016, USA

    Kevin Ryan

Authors
  1. Mihwa Na
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  2. Susana T. Valente
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  3. Kevin Ryan
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Correspondence to Kevin Ryan .

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Editors and Affiliations

  1. Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA

    Narendra Wajapeyee

  2. Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA

    Romi Gupta

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Na, M., Valente, S.T., Ryan, K. (2017). Optimizing In Vitro Pre-mRNA 3′ Cleavage Efficiency: Reconstitution from Anion-Exchange Separated HeLa Cleavage Factors and from Adherent HeLa Cell Nuclear Extract. In: Wajapeyee, N., Gupta, R. (eds) Eukaryotic Transcriptional and Post-Transcriptional Gene Expression Regulation. Methods in Molecular Biology, vol 1507. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6518-2_14

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  • DOI: https://doi.org/10.1007/978-1-4939-6518-2_14

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6516-8

  • Online ISBN: 978-1-4939-6518-2

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