Scripps VIVO scripps research logo

  • Index
  • Log in
  • Home
  • People
  • Organizations
  • Research
  • Events
Search form

Analysis of RNA processing reactions using cell free systems: 3' end cleavage of pre-mRNA substrates in vitro

Academic Article
uri icon
  • Overview
  • Research
  • Identity
  • Additional Document Info
  • View All
scroll to property group menus

Overview

authors

  • Jablonski, J.
  • Clementz, M.
  • Ryan, K.
  • Valente, Susana Tereno

publication date

  • May 2014

journal

  • Journal of Visualized Experiments : JoVE  Journal

abstract

  • The 3' end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages. Processing of the 3' end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5' cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3' end processing of HIV-1 mRNAs.

subject areas

  • Autoradiography
  • Cell-Free System
  • Electrophoresis
  • HIV-1
  • Humans
  • Phosphorus Radioisotopes
  • RNA Polymerase II
  • RNA Precursors
  • RNA, Messenger
scroll to property group menus

Research

keywords

  • 3' Processing Complex
  • Cleavage
  • Infectious Diseases
  • Issue 87
  • Nuclear extracts
  • Polyadenylation
  • mRNA processing
scroll to property group menus

Identity

PubMed Central ID

  • PMC4030693

International Standard Serial Number (ISSN)

  • 1940-087X

Digital Object Identifier (DOI)

  • 10.3791/51309

PubMed ID

  • 24835792
scroll to property group menus

Additional Document Info

start page

  • e51309

issue

  • 87

©2021 The Scripps Research Institute | Terms of Use | Powered by VIVO

  • About
  • Contact Us
  • Support