Genome-wide analysis of RNA polymerase II termination at protein-coding genes. Saccharomyces cerevisiae

At the end of protein-coding genes, RNA polymerase (Pol) II undergoes a concerted transition that involves 3’-processing of the pre-mRNA and transcription termination. Here we conduct a genome-wide analysis of this 3’-transition in yeast. We find that the 3’-transition globally requires the Pol II elongation factor Spt5 and factors involved in the recognition of the poly-adenylation (pA) site and in endonucleolytic RNA cleavage. Pol II release from DNA occurs in a narrow termination window downstream of the pA site and generally requires the ‘torpedo’ exonuclease Rat1 (human XRN2). The Rat1-interacting factor Rai1 contributes to RNA degradation downstream of the pA site. Defects in the 3’-transition result in transcription interference at downstream genes, which is attenuated by a back-up termination mechanism. Overall design: In this study, we depleted the main termination factors of the Rat1–Rai1–Rtt103 exonuclease complex as well as the CFIA factor Pcf11 from the nucleus and monitored changes in RNA synthesis using the anchor-away (aa) technique and 4-thiouracil (4tU) sequencing, respectively. We applied ChIP-seq and PAR-CLIP to obtain high-confidence genome and transcriptome maps, respectively. Additionally, we assayed the effects after nuclear depletion of Spt4/5 as well as for Spt5 mutants lacking both the C-terminal repeat (CTR) and fifth KOW domain. We show that Rat1 is needed for termination of the transcribing RNA Polymerase (Pol) II by comparing the ChIP-seq profiles of Rpb1 before (Rat1no) and after nuclear depletion (Rat1aa) of the exonculease Rat1.