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Updated: Nov 15, 2025

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro
Published on: May 3, 2014
In vitro RNA Cleavage Assays to Characterize IRE1-dependent RNA Decay
G Elif Karagöz1, Jirka Peschek2,3, Peter Walter2,3
1Max Perutz Labs Vienna, Medical University of Vienna, Vienna, Austria.
Abstract:
The kinase/RNase IRE1 is a key effector of the cellular response to endoplasmic reticulum stress. The RNase activity of IRE1 can be measured in cells or in the test tube. Here we describe a protocol for the in vitro cleavage and analysis of RNA substrates of IRE1. The method consists of the in vitro transcription, purification and re-folding of IRE1 substrate RNAs followed by their cleavage using recombinant cytosolic kinase/RNase domains of IRE1 and the separation of the resulting fragments by denaturing polyacrylamide gel electrophoresis. This protocol allows the study of the cleavage kinetics of IRE1's RNA substrates in vitro.
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