Here, we identified key signatures of transcriptional termination mechanisms that are altered by heat shock, enabling global readthrough of the 3′ends of mRNA genes. The Pol II 3′ pause is lost, CTD phosphorylation at Ser2 and Tyr1 changes, and endonucleolytic cleavage of the transcript is impaired, which is relieved by expression of RBBP6, a cleavage activator. Our data support a multifaceted mechanism of readthrough during stress, whereby changes to both Pol II and the termination machinery trigger transcription to bypass normal termination sites.
The stress-induced readthrough we observed was pervasive, with 85% of the analyzable genes showing downstream transcription after heat shock. There was no correlation with the level of transcription or activation/repression and no indication of any gene class specificity. Prior work has shown that genes with readthrough are largely overlapping between types of cellular stress.19 Moreover, only loose correlations have been found between sequence markers for termination, such as the strength of the PAS and/or flanking GA-rich regions, and readthrough transcription.26 Distinguishing characteristics of readthrough susceptibility for any given stress remain to be elucidated.
Our Pol II ChIP-seq data show that heat shock induces global loss of 3′ Pol II pausing. This occurred at genes with and without readthrough, indicating that lack of Pol II pausing is not sufficient to cause readthrough. This raises interesting questions about the relationship between Pol II deceleration and transcription termination mechanisms. Current models suggest the slowing of Pol II helps the 5′-to-3′ exonuclease XRN2 to degrade the nascent RNA, catch up to transcribing Pol II, and trigger the dissociation of the polymerase from the DNA.9,10,34 This model requires that nascent RNA cleavage occurs to generate the 5′ end for XRN2 to bind. For readthrough genes that retain RNA cleavage during heat shock, the failure of Pol II to decelerate could prevent XRN2 from “catching up,” thereby enabling downstream transcription. For readthrough genes that lose cleavage, Pol II deceleration likely enhances readthrough by having a fast-moving polymerase.
