Analysis of RNA polymerase II ubiquitylation and proteasomal degradation

• Published in: Methods (San Diego, Calif.) Vol. 159-160; pp. 146 - 156
• Main Authors: Tufegdzic Vidakovic, Ana, Harreman, Michelle, Dirac-Svejstrup, A. Barbara, Boeing, Stefan, Roy, Anindya, Encheva, Vesela, Neumann, Michelle, Wilson, Marcus, Snijders, Ambrosius P., Svejstrup, Jesper Q.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.04.2019; Academic Press
• Subjects: Animals; Degradation; DNA Damage; Humans; Mammals - genetics; Mammals - metabolism; Proteasome Endopeptidase Complex - metabolism; Proteolysis; RNA polymerase II; RNA Polymerase II - analysis; RNA Polymerase II - antagonists & inhibitors; RNA Polymerase II - genetics; RNA Polymerase II - metabolism; Transcription, Genetic; Ubiquitination; Ubiquitylation; Ultraviolet Rays; UV-irradiation; Yeasts - enzymology; Yeasts - genetics; Yeasts - metabolism; Degradation; UV-irradiation; Ubiquitylation; RNA polymerase II
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2019.02.005

•A variety of stimuli, including UV-irradiation, cause RNAPII ubiquitylation and degradation.•Specific considerations are required for reproducible UV-irradiation of human and yeast cells.•Dsk2/MultiDsk pulldown provides robust enrichment of ubiquitylated protein species.•RNAPII degradation level depends on multiple variables. 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.