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In Vitro System for Coupling RNAP II Transcription to Primary microRNA Processing and a Three-Way System for RNAP II Transcription/Splicing/microRNA Processing

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miRNA Biogenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1823))

Abstract

In the genome, primary microRNAs (pri-miRNAs) are encoded either as independent transcriptional units with their own promoters (intergenic miRNAs) or within the introns of other genes (intronic miRNAs). Here, we report two methods, one that we established for coupled RNAP II transcription and pri-miRNA processing and the other that is a three-way system for RNAP II transcription, pri-miRNA processing, and pre-mRNA splicing. In these systems, CMV-DNA constructs encoding the processing substrates are incubated in HeLa cell nuclear extracts in the presence of 32P-UTP to generate the nascent RNAP II transcripts, which are processed efficiently by the endogenous RNA processing machineries in nuclear extracts.

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Acknowledgments

We are grateful to members of the Reed lab for comments on the manuscript. This work was supported by an NIH grant GM122524 to RR.

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Authors and Affiliations

  1. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Shanye Yin, Alexander Iocolano, Yong Yu, Jaya Gangopadhyay & Robin Reed

Authors
  1. Shanye Yin
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  2. Alexander Iocolano
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  3. Yong Yu
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  4. Jaya Gangopadhyay
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  5. Robin Reed
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Corresponding author

Correspondence to Robin Reed .

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Editors and Affiliations

  1. Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark

    Ulf Andersson Vang Ørom

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Yin, S., Iocolano, A., Yu, Y., Gangopadhyay, J., Reed, R. (2018). In Vitro System for Coupling RNAP II Transcription to Primary microRNA Processing and a Three-Way System for RNAP II Transcription/Splicing/microRNA Processing. In: Ørom, U. (eds) miRNA Biogenesis. Methods in Molecular Biology, vol 1823. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-8624-8_4

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  • DOI: https://doi.org/10.1007/978-1-4939-8624-8_4

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  • Publisher Name: Springer, New York, NY

  • Print ISBN: 978-1-4939-8623-1

  • Online ISBN: 978-1-4939-8624-8

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