Ubiquitylation of Pol II controls early stages of the transcription cycle [TT-seq]

Control of RNA Polymerase II (Pol II) through ubiquitylation is essential for the DNA-damage response. Here we reveal a distinct ubiquitylation pathway in human cells that targets excessive and defective Pol II molecules at the initial stages of the transcription cycle. This pathway, mediated by ARMC5CUL3 ubiquitin ligase, drives homeostatic Pol II turnover, and is further enhanced when early stages of transcription - initiation and pausing - are perturbed. Upon ARMC5 loss, Pol II accumulates in the free pool and in the promoter-proximal zone, but is not permitted into elongation. We identify Integrator subunit 8 (INTS8) as a gatekeeper preventing the release of excess Pol II molecules into gene bodies. Combined loss of ARMC5 and INTS8 has strong detrimental effects on cell growth, with ARMC5 loss exacerbating transcriptional defects seen in INTS8-depleted cells. These findings uncover that ARMC5CUL3 and INTS8 form a collaborative checkpoint, monitoring the quantity and quality of transcription complexes before they are licensed into elongation. To study how ARMC5 loss confers partial resistance to XBP inhibition by triptolide we generated ARMC5 KO cells in HEK293 Trex flp In background. We treated WT and ARMC5 KO cells with triptolide at 300 nM for 2 h. 4SU was added 15 minutes prior to harvesting in all samples. To study the function of INTS8 on the ARMC5 KO background we depleted INTS8 with siRNA targeting INTS8 48 h prior to triptolide treatment.