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Timing RNA polymerase pausing with TV-PRO-seq.

Jie Zhang1, Massimo Cavallaro1,2, Daniel Hebenstreit1

  • 1School of Life Sciences, Gibbet Hill Campus, the University of Warwick, CV4 7AL Coventry, UK.

Cell Reports Methods
|November 1, 2021
PubMed
Summary
This summary is machine-generated.

Scientists developed a new method to measure how long transcription polymerases pause genome-wide. This reveals polymerase pausing is more frequent but shorter near promoters, and longer for highly expressed genes.

Keywords:
H3K36me3NELFPRO-seqRNA polymerasegene expressionnext-generation sequencingpolymerase pausingpromoter-proximal pausingtranscription dynamicstranscriptional noise

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Area of Science:

  • Molecular Biology
  • Genomics
  • Gene Regulation

Background:

  • Polymerase pausing, a transient stop of transcriptionally engaged polymerases, is a known regulatory mechanism in metazoan gene transcription.
  • Pausing predominantly occurs in promoter-proximal regions, but a comprehensive, high-resolution understanding of its dynamics remains elusive.
  • A lack of genome-wide measurements at high resolution has hindered detailed analysis of polymerase pausing times.

Purpose of the Study:

  • To develop and validate a novel assay for genome-wide measurement of polymerase pausing times at single-base resolution.
  • To investigate the characteristics of polymerase pausing in human cells, particularly in promoter-proximal regions.
  • To correlate polymerase pausing dynamics with gene expression levels and transcriptional noise.

Main Methods:

  • Development of the time-variant precision nuclear run-on and sequencing (TV-PRO-seq) assay, an advancement of the standard PRO-seq technique.
  • Application of TV-PRO-seq to human cells to achieve genome-wide estimation of pausing times.
  • Integration of TV-PRO-seq data with single-cell gene expression data and histone modification analysis.

Main Results:

  • TV-PRO-seq successfully estimated genome-wide polymerase pausing times at single-base resolution.
  • Polymerases exhibit higher pausing frequencies but shorter pausing durations in promoter-proximal regions compared to other genomic locations.
  • Longer polymerase pausing times correlate with highly expressed genes, while increased pausing frequency and slightly longer pausing times are associated with transcriptionally noisy genes.
  • The histone modification marker H3K36me3 shows a relationship with polymerase pausing.

Conclusions:

  • The TV-PRO-seq assay provides a powerful tool for high-resolution, genome-wide analysis of polymerase pausing dynamics.
  • Polymerase pausing exhibits distinct temporal and spatial characteristics across the genome, influencing gene expression variability.
  • Histone modifications, such as H3K36me3, may play a role in modulating polymerase pausing.