Protocol for rapidly inducing genome-wide RNA Pol II hyperphosphorylation by selectively disrupting INTAC phosphatase activity

Yu-Xin Ji; Shibin Hu; Fei Xavier Chen

doi:10.1016/j.xpro.2023.102640

Protocol for rapidly inducing genome-wide RNA Pol II hyperphosphorylation by selectively disrupting INTAC phosphatase activity

STAR Protoc. 2023 Dec 15;4(4):102640. doi: 10.1016/j.xpro.2023.102640. Epub 2023 Oct 12.

Authors

Yu-Xin Ji¹, Shibin Hu², Fei Xavier Chen³

Affiliations

¹ Fudan University Shanghai Cancer Center, Shanghai Key Laboratory of Medical Epigenetics, Shanghai Key Laboratory of Radiation Oncology, Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
² Fudan University Shanghai Cancer Center, Shanghai Key Laboratory of Medical Epigenetics, Shanghai Key Laboratory of Radiation Oncology, Institutes of Biomedical Sciences, Fudan University, Shanghai, China. Electronic address: shibinhu@fudan.edu.cn.
³ Fudan University Shanghai Cancer Center, Shanghai Key Laboratory of Medical Epigenetics, Shanghai Key Laboratory of Radiation Oncology, Institutes of Biomedical Sciences, Fudan University, Shanghai, China. Electronic address: feixchen@fudan.edu.cn.

Abstract

While several inhibitors targeting RNA polymerase II (Pol II) kinases have been applied for inhibiting RNA Pol II phosphorylation, there are few approaches for inducing RNA Pol II hyperphosphorylation. Here, we present a protocol for constructing the INTS8 degradation tag (dTAG) system combined with ectopic expression of N-terminally truncated INTS8 (INTS8-ΔN) in DLD-1 cells. We describe steps for INTS8-dTAG cell line construction, validation of knockin and degradation, and INTS8-ΔN rescue. We then detail validation of RNA Pol II phosphorylation upregulation. For complete details on the use and execution of this protocol, please refer to Hu et al. (2023).¹.

Keywords: Cell Biology; Gene Expression; Molecular Biology; Protein Biochemistry.

MeSH terms

Cell Line
Phosphoric Monoester Hydrolases / genetics
Phosphorylation
RNA Polymerase II* / genetics
RNA Polymerase II* / metabolism
Transcription, Genetic*

Substances

RNA Polymerase II
Phosphoric Monoester Hydrolases