Timing RNA polymerase pausing with TV-PRO-seq

ABSTRACT: Summary Transcription of many genes in metazoans is subject to polymerase pausing, which is the transient stop of transcriptionally engaged polymerases. This is known to mainly occur in promoter-proximal regions but it is not well understood. In particular, a genome-wide measurement of pausing times at high resolution has been lacking. We present here the time-variant precision nuclear run-on and sequencing (TV-PRO-seq) assay, an extension of the standard PRO-seq that allows us to estimate genome-wide pausing times at single-base resolution. Its application to human cells demonstrates that, proximal to promoters, polymerases pause more frequently but for shorter times than in other genomic regions. Comparison with single-cell gene expression data reveals that the polymerase pausing times are longer in highly expressed genes, while transcriptionally noisier genes have higher pausing frequencies and slightly longer pausing times. Analyses of histone modifications suggest that the marker H3K36me3 is related to the polymerase pausing. Graphical abstract Highlights • Measurement of polymerase pausing times genome-wide at single-base resolution• Promoter-proximal regions have more frequent but shorter individual pausing events• Genes with high transcriptional noise have more and longer pausing• H3K36me3 correlates with pausing Motivation Various next-generation sequencing-based methods, including Pol II ChIP-seq, GRO-seq, NET-seq, and PRO-seq, have been able to reveal the presence of polymerase pausing. However, these methods are mainly based on detection of polymerase occupancy, which is affected by factors other than the pausing. As an example, recent research revealed that polymerase has a high abortive transcription rate, especially at genes featuring strong pausing, which also contributes to high polymerase occupancy in the promoter-proximal regions. Further, the accuracy of the most widely used method to measure pausing—treating cells with triptolide (Trp)—is challenged by the latter's slow uptake. A method that outputs the actual pausing time is still required to dissect the pausing profile. We have developed TV-PRO-seq, which reveals polymerase pausing times at single-base resolution genome-wide and is not influenced by the polymerase turnover and other confounding factors. Zhang et al. develop a next-generation sequencing method based on PRO-seq that allows genome-wide measurement of pausing times of RNA polymerases at single-base resolution. TV-PRO-seq reveals frequent short pausing events in promoter-proximal regions and uncovers links between pausing times and gene expression as well as chromatin states.

SUBMITTER: Zhang J 

PROVIDER: S-EPMC8547241 | biostudies-literature |

REPOSITORIES: biostudies-literature