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Methods. 2019 Apr 15;159-160:146-156. doi: 10.1016/j.ymeth.2019.02.005. Epub 2019 Feb 13.

Analysis of RNA polymerase II ubiquitylation and proteasomal degradation.

Author information

1
Mechanisms of Transcription Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
2
Protein Analysis and Proteomics Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK.
3
Mechanisms of Transcription Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. Electronic address: jesper.svejstrup@crick.ac.uk.

Abstract

Transcribing RNA polymerase II (RNAPII) is decorated by a plethora of post-translational modifications that mark different stages of transcription. One important modification is RNAPII ubiquitylation, which occurs in response to numerous different stimuli that cause RNAPII stalling, such as DNA damaging agents, RNAPII inhibitors, or depletion of the nucleotide pool. Stalled RNAPII triggers a so-called "last resort pathway", which involves RNAPII poly-ubiquitylation and proteasome-mediated degradation. Different approaches have been described to study RNAPII poly-ubiquitylation and degradation, each method with its own advantages and caveats. Here, we describe optimised strategies for detecting ubiquitylated RNAPII and studying its degradation, but these protocols are suitable for studying other ubiquitylated proteins as well.

KEYWORDS:

Degradation; RNA polymerase II; UV-irradiation; Ubiquitylation

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