A unified allosteric/torpedo mechanism for transcriptional termination on human protein-coding genes

The so-called allosteric and torpedo models have been used for the past thirty years to explain how transcription terminates on protein-coding genes. The former invokes conformational changes in the transcription complex and the latter involves degradation of the downstream product of poly(A) signal (PAS) processing. Here, we describe a single mechanism incorporating features of both models. We show that CPSF73 is indispensable for transcriptional termination on protein-coding and its loss causes profound read-through genome-wide. CPSF73 functions upstream of allosteric modifications to the elongation complex that cause Pol II to slow down after the PAS. This state is enriched by rapid depletion of XRN2 and promoted by protein phosphatase 1 (PP1), the inhibition of which confers runaway read-through in the absence of XRN2. These allosteric changes facilitate XRN2-dependent termination, by aiding its capture of Pol II, rather than constituting a termination pathway in themselves. Our experiments unify the long-standing allosteric and torpedo models for transcriptional termination. Chromatin-associated and nuclear RNA-seq was performed on a CPSF73 tagged auxin-inducible degron (AID) cell line treated with or without auxin to induce rapid depletion of the CPSF73 protein. The AID cassette consists of an in-frame insertion of the AID tag at the 3' exon, P2A cleavage sequence, antibiotic resistance marker and SV40 polyA site. Two repair plasmids containing either Hygromycin or Neomycin resistance markers were used to select for biallelic insertions of the repair cassettes by CRISPR/Cas9. This HCT116 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. The TIR1 protein is the E3 ubiquitin ligase required to induce degradation of AID containing proteins in the presence auxin.