Nascent transcript folding plays a major role in determining RNA polymerase elongation rates

Abstract

HIGHLIGHTSStructures in the nascent RNA correlate with rapid elongation by RNAPI in vivo Stable RNA structures limit RNAPI backtracking in vitro GC content in the transcription bubble tunes transcription elongation rate Nascent transcript folding modulates dynamics of all three RNAPs in vivo ABSTRACT Transcription elongation rates are important for RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S.cerevisiae.Analysis of Miller chromatin spreads and mapping RNAPI using UV crosslinking, revealed a marked 5' bias and strikingly uneven local polymerase occupancy, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution; folding energy and G+C-content. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high G+C within the transcription bubble slows elongation. We developed a mathematical model for RNAPI elongation, which confirmed the importance of nascent RNA folding in transcription. RNAPI from S.pombe was similarly sensitive to transcript folding, as were S.cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site usage indicating regulatory significance for transcript folding.(150 words -150 words max)

Profiling of Nascent Lariat Intermediates Reveals Key Genetic Determinants of the Timing of Human Co-transcriptional Splicing

“…High GC content likely impacts splicing timing in multiple ways, such as weakening splice site strength (Amit et al, 2012), stabilizing local RNA secondary structure (Fig. S5H), accelerating or decelerating transcription (Saldi et al, 2021;Turowski et al, 2020;Veloso et al, 2014;Zamft et al, 2012), and associating with different regions of the nucleus, given the impact of GC content on the conformation of the genome (Lemaire et al, 2019;van Steensel and Belmont, 2017). Consistent with an impact on RNA structure and splice site recognition, GC-rich cassette exons are skipped after knockdown of the RNA helicases DDX5 and DDX17, which promote U1 snRNP binding (Lemaire et al, 2019).…”