RNA Cleavage Assays to Characterize IRE1-dependent RNA Decay.

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 cleavage and analysis of RNA substrates of IRE1. The method consists of the 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 .