In vitro system for coupling RNAP II transcription to primary microRNA processing and a three-way system for RNAP II transcription/splicing/microRNA processing

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

Research output: Chapter in Book/Report/Conference proceedingChapter

1 Scopus citations

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 32 P-UTP to generate the nascent RNAP II transcripts, which are processed efficiently by the endogenous RNA processing machineries in nuclear extracts.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages43-50
Number of pages8
DOIs
StatePublished - 2018
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1823
ISSN (Print)1064-3745

Keywords

  • Coupled steps in gene expression
  • Pre-mRNA splicing
  • Primary microRNA processing
  • RNAP II transcription

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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