CPSF73 activation and 3′ RNA polymerase II pausing are lost during readthrough transcription after heat shock

Environmental stresses induce RNA polymerase II (Pol II) to read through normal termination sites at mRNA genes using unknown mechanisms. We observe strong readthrough transcription downstream of thousands of genes after heat shock. Additionally, Pol II no longer pauses after the 3' ends of genes. Heat shock also increases phosphorylation of Tyr1 and Ser2 residues in the Pol II C-terminal domain (CTD) at the 3' ends of genes, which is attenuated at genes with readthrough transcription. Endonucleolytic cleavage of the nascent transcript, key to normal termination, is defective at readthrough genes after heat shock. However, CPSF73, the endonuclease responsible, remains present. Overexpressing an activator of CPSF73, RBBP6, during heat shock rescues the loss of cleavage and dampens readthrough transcription. Together, our results show that heat shock alters Pol II speed, CTD phosphorylation, and CPSF73 activity via RBBP6, contributing to a multifaceted mechanism that enables readthrough of termination sites during cellular stress.