Analysis of RNA polymerase II ubiquitylation and proteasomal degradation

Ana Tufegdzic Vidakovic, Michelle Harreman, A. Barbara Dirac-Svejstrup, Stefan Boeing, Anindya Roy, Vesela Encheva, Michelle Neumann, Marcus Wilson, Ambrosius P. Snijders, Jesper Q. Svejstrup*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)146-156
Number of pages11
JournalMethods
Volume159-160
Early online date13 Feb 2019
DOIs
Publication statusE-pub ahead of print - 13 Feb 2019

Keywords

  • degradation
  • RNA polymerase II
  • ubiquitylation
  • UV-irradiation

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