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

• Published in: STAR protocols 2023-12, Vol.4 (4), p.102640-102640, Article 102640
• Main Authors: Ji, Yu-Xin, Hu, Shibin, Chen, Fei Xavier
• Format: Article
• Language: English
• Subjects: Cell Biology; Cell Line; Gene Expression; Molecular Biology; Phosphoric Monoester Hydrolases - genetics; Phosphorylation; Protein Biochemistry; Protocol; RNA Polymerase II - genetics; RNA Polymerase II - metabolism; Transcription, Genetic
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102640

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 [Display omitted] •A platform for rapidly and specifically inducing RNA Pol II hyperphosphorylation•Create INTS8-dTAG+ΔN cell line to target INTAC phosphatase without affecting PP2A•Applicable to studying the direct functions of Pol II phosphorylation in editable cell lines Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.