The role of INTS3 in transcriptional regulation

Integrator (INT) is an RNA polymerase II-associated transcription complex, comprising fifteen subunits clustered into distinct functional modules. Each module individually contributes to Integrator’s various regulatory roles in transcription which include modulation of RNA Pol II (RNAPII) pause-release transition, endonucleolytic cleavage and processing of nascent RNA species, targeted dephosphorylation of basal transcriptional machinery, and DNA repair. In particular, Integrator subunit 3 (INTS3) has been implicated in DNA damage repair in conjunction with the Sensor of ssDNA (SOSS) complex, alongside a role in preventing reassociation of RNAPII following premature transcriptional termination. However, a satisfactory model of the mechanism by which INTS3 contributes to overall transcriptional regulation still remains elusive. In this light, a dTAG targeted protein degradation (TPD) system for INTS3 was generated in OVCAR8 cells to probe the rapid effects of INTS3 ablation on transcriptional regulation. The degradation of INTS3 is observed to upregulate the expression of various INT subunits and phosphoserine states of Ser2, Ser5 and Ser7 of the carboxyterminal domain (CTD) of RNAPII, and additionally may have an effect on INTAC (INT conjugated with PP2A) complex formation. Furthermore, INTS3 ablation is correlated with changes to the transcriptome with respect to the upregulation of immediate early response genes (IERGs). In a wider context, SOSS complex constituents were found to colocalize with INTS3 across the genome, predominantly at the AP-1 promoter element which is responsible for IERG expression. Moreover, it was observed that splicing factor 3 (SF3) subunits are differentially pulled down dependent on INTS3 ablation. Lastly, INTS3 degradation results in the reduction of RNAPII across the genome alongside an increase in pSer2 and pSer5 states, as well as a more efficient release of RNAPII into the gene body indicated by a decreased traveling ratio for RNAPII.