Splicing Kinetics and Coordination Revealed by Direct Nascent RNA Sequencing through Nanopores

Drexler, Heather L.; Choquet, Karine; Churchman, L. Stirling

doi:10.1016/j.molcel.2019.11.017

Cited by 165 publications

(230 citation statements)

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“…The mRNA transcribed by most eukaryotic genes are spliced, a process in which the intronic RNA sequence is removed and the exonic RNA are joined together to form the ultimate mature mRNA sequence. A major question in the field is how tightly associated the process of transcription and splicing are, with some work suggesting that splicing occurs very shortly after the RNA polymerase transcribes a particular splice junction (Beyer et al, 1981;Carrillo Oesterreich et al, 2016) , while other work suggests that many pre-mRNAs are fully transcribed before splicing occurs (Coulon et al, 2014;Drexler et al, 2019;Tsai et al, 1980) . The relative spatial locations of nascent pre-mRNA, fully transcribed pre-mRNA, and mature mRNA species have the potential to directly reveal where-and consequently in what order-the processes of transcription and splicing occur.…”

Section: Introductionmentioning

confidence: 99%

“…In lieu of direct visualization, many have utilized biochemical fractionation to infer the location of various intermediates (Bhatt et al, 2012;Drexler et al, 2019;Mayer et al, 2015;Pandya-Jones and Black, 2009;Pandya-Jones et al, 2013;Tilgner et al, 2012;Wuarin and Schibler, 1994) . Fractionation methods separate cellular RNA into different compartments, such as the putatively chromatin associated RNA, nucleoplasmic RNA, and cytoplasmic RNA, by centrifuging the cellular components in different lysis buffers and sedimentation gradients (Mayer and Churchman, 2017;Wuarin and Schibler, 1994) .…”

Section: Introductionmentioning

confidence: 99%

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Post-transcriptional splicing can occur in a slow-moving zone around the gene

Coté

Bayatpour

et al. 2020

Preprint

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Splicing is the molecular process by which introns are removed from pre-mRNA and exons are joined together to form the sequence of the mature mRNA. Measuring the timing of splicing relative to the transcription of nascent RNA has yielded conflicting interpretations. Biochemical fractionation suggests that RNA is spliced primarily during the process of transcription, but imaging of nascent RNA suggests that splicing happens after the process of transcription has been completed. We use single molecule RNA FISH together with expansion microscopy to measure the spatial distribution of nascent and partially spliced transcripts in mammalian cells, allowing us to infer the delay between when an intron is transcribed and when it is spliced out of a pre-mRNA. We show that 4 out of 4 genes we interrogated exhibit some post-transcriptional splicing, and that introns can be spliced in any order. We also show that completely synthesized RNA move slowly through a transcription site proximal zone while they undergo additional splicing and potentially other processing after transcription is completed. In addition, upon leaving this zone, some genes' transcripts localize to speckles during the process of splicing but some appear to traffic freely through the nucleus without localizing to any other nuclear compartment. Taken together, our observations suggest that the regulation of the timing and localization of splicing is specific to individual introns, as opposed to the previously surmised immediate excision of introns after transcription.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Post-transcriptional splicing can occur in a slow-moving zone around the gene

Coté

Bayatpour

et al. 2020

Preprint

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“…This is important, because many RNA processing factors act very quickly on the nascent transcript. For example, splicing of pre-mRNA is strikingly speedy in yeast (Wallace and Beggs, 2017), although more heterogeneous in metazoans (Alpert et al, 2017;Drexler et al, 2019), and this can shape the mature mRNA through alternative splicing (Saldi et al, 2016). Understanding the detailed kinetics of transcription elongation in vivo will therefore be predictive for processing decisions.…”

Section: Discussionmentioning

confidence: 99%

“…We speculate that slower elongation of RNAPII facilitates splicing by allowing more time for recognition of the 3' SS by splicing factors associated with C-terminal domain of the polymerase. Notably, the "window of opportunity" for 3' SS recognition is presumably substantially shorter than the actual pre-mRNA splicing reaction that is assessed by transcript sequencing in yeast and mammalian systems (Alpert et al, 2017;Drexler et al, 2019;Neugebauer, 2019;Wachutka et al, 2019;Wallace and Beggs, 2017).…”

Section: Discussionmentioning

confidence: 99%

“…The in vivo distribution of RNAPI was initially analyzed using Miller chromatin spreads visualized by electron microscopy, in yeast and many other organisms (for an example see (Osheim et al, 2009)). Subsequently, polymerase distributions have been mapped using techniques that include ChIP, NET-seq, CRAC and metabolic labelling (Booth et al, 2016;Churchman and Weissman, 2011;Clarke et al, 2018;Drexler et al, 2019;Mayer et al, 2015;Milligan et al, 2016;Nojima et al, 2015;Schwalb et al, 2016;Turowski et al, 2016;Vinayachandran et al, 2018). Commonly, DNA or RNA is recovered in association with the polymerase and identified by sequencing.…”

Section: Introductionmentioning

confidence: 99%

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

Turowski

Petfalski

Goddard

et al. 2020

Preprint

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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)

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Speculating on the Roles of Nuclear Speckles: How RNA‐Protein Nuclear Assemblies Affect Gene Expression

Hasenson

Shav‐Tal

2020

BioEssays

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Nuclear speckles are eukaryotic nuclear bodies enriched in splicing factors. Their exact purpose has been a matter of debate. The different proposed roles of nuclear speckles are reviewed and an additional layer of function is put forward, suggesting that by accumulating splicing factors within them, nuclear speckles can buffer the nucleoplasmic levels of splicing factors available for splicing and thereby modulate splicing rates. These findings build on the already established model that nuclear speckles function as a storage/recycling site for splicing factors. Many studies have demonstrated proximity between nuclear speckles and sites of active transcription, suggesting that this juxtaposition can enhance the rates of gene expression. It is found that nuclear speckle disassembly increases splicing factor availability in the nucleoplasm, leading to an increase in splicing rates and faster release of nascent transcripts from the gene after transcription. Altogether, this era in which genomic and imaging approaches are applied to study nuclear organization has expanded the outlook on the possible roles of nuclear speckles.

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Splicing Kinetics and Coordination Revealed by Direct Nascent RNA Sequencing through Nanopores

Cited by 165 publications

References 91 publications

Post-transcriptional splicing can occur in a slow-moving zone around the gene

Post-transcriptional splicing can occur in a slow-moving zone around the gene

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

Speculating on the Roles of Nuclear Speckles: How RNA‐Protein Nuclear Assemblies Affect Gene Expression

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