Tripartite phosphorylation of SPT5 by CDK9 times pause release and tunes elongation rate of RNA polymerase II (CUT&RUN)

The RNA polymerase II (RNAPII) transcription cycle is regulated throughout its duration by protein phosphorylation. Previously, two regions phosphorylated by cyclin-dependent kinase 9 (CDK9) in elongation factor SPT5—the linker between Kyrpides-Ouzounis-Woese (KOW) x-4 and 5 domains and carboxy-terminal repeat (CTR) 1—were implicated in promoter-proximal pausing and termination, respectively. Here, we show that phosphorylations in the linker, CTR1, and a third region, CTR2, coordinately control pause release, elongation speed, and termination in HCT116 human colon cancer cells. Pausing was unaffected or increased by mutations preventing CTR1 or CTR2 phosphorylation, respectively, but attenuated when both CTRs were mutated. Whereas loss of CTR1 phosphorylation slowed elongation and repressed nascent transcription, simultaneous CTR2 mutation partially reversed both effects. Nevertheless, mutating both CTRs had additive effects on splicing, termination, steady-state mRNA levels, and cell proliferation. Therefore, tripartite SPT5 phosphorylation times pause release and tunes RNAPII elongation rate to ensure productive transcription and cell viability. The effects of CTR1 and CTR2 mutations on elongation index suggested complex regulation of RNAPII elongation velocity by CTR phosphorylation. To measure elongation rates directly in cells dependent on different SPT5 variants, we reversibly blocked RNAPII in the promoter-proximal region by a 3-hr treatment with the CDK inhibitor 5,6-dichloro-1-b-D-ribofuranosyl-benzimidazole (DRB), and monitored progression of the transcribing RNAPII wavefront by CUT&RUN at 0, 3 and 5 min after DRB washout.