An allosteric/intrinsic mechanism supports termination of snRNA transcription.

Termination of protein-coding gene transcription by RNA polymerase II (Pol II) depends on endonucleolytic cleavage at the poly(A) site and the activity of a 5'->3' “torpedo” exoribonuclease. Other Pol II transcripts also undergo endonucleolytic cleavage suggesting common themes for its termination. Nevertheless, many RNA polymerases employ intrinsic/allosteric termination that directly defines transcript 3' ends. We have analysed termination of snRNA transcription in humans, which utilises the endoribonucleolytic integrator complex. Integrator is involved, but termination continues after its depletion. This implicates additional mechanisms and shows that endo- and 5'->3' exonuclease activities are not necessarily a basis for all Pol II termination. Although the alternative process acts as a failsafe in the absence of integrator it contributes significantly to snRNA termination in the natural situation. Long-read sequencing reveals its products to have stochastic 3' ends that are terminated before integrator cleavage, ruling out 5'->3' exonuclease contribution(s) and supporting an allosteric/intrinsic mechanism. Termination of some snRNA transcription occurs at T-runs further indicating common principles between this mechanism and those used by other RNA polymerases. Overall design: Nuclear and sub-nuclear selected RNA was isolated auxin-indubile degron (AID) tagged HCT116 cell lines treated with or without auxin, and sequenced using a combination of library approaches and short- or long-read technologies to study the termination of snRNA gene transcription temination. RNA Polymrease II associated DNA fragmnts were also analysed in the form of a ChIP-Seq experiment. We integrated an in-frame insertion of AID tag, P2A cleavage site, antibiotic resistance marker and SV40 polyA site to the 3' end of three proteins: DIS3-AID, CPSF73 or FUS. Modified DIS3-AID and FUS-AID cells also ectopically expressed TIR1 from a sleeping beauty transposon integrated cassette. The CPSF73-AID cell line was additionally modified at the AAVS1 safe-habour locus by ectopic integration of a doxycycline-inducible TIR1 gene using a pMK243 repair template plasmid (addgene #72835) via CRISPR/Cas9. In all cell lines, TIR1 protein is the E3 ubiquitin ligase required to induce degradation of AID containing proteins in the presence auxin however, CPSF73-AID cells were not treated with auxin but were instead used to RNAi deplete the INTS1 protein. Using a similar approach, we replaced the AID tag repair cassette with a Small Molecule-Assisted Shutoff (SMASh) inducible protease degron tag which was the inserted at the 3'-end of INTS11 to induce protein depletion (isolated from pCS6-YFP-SMASh (Addgene Plasmid #68853). Nuclear RNA was extracted from INTS11-SMASh cells treated with asunaprevir or not.