RNA polymerase II stalling at pre-mRNA splice sites is enforced by ubiquitination of the catalytic subunit

Milligan, Laura; Sayou, Camille; Tuck, Alex; Auchynnikava, Tatsiana; Reid, Jane E. A.; Alexander, Ross D.; Alves, Flávia de Lima; Allshire, Robin C.; Spanos, Christos; Rappsilber, Juri; Beggs, Jean D.; Kudla, Grzegorz; Tollervey, David

doi:10.7554/elife.27082

Cited by 18 publications

(20 citation statements)

References 70 publications

(117 reference statements)

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“…Similarly, cellular concentration of polyadenylation factors has been linked to coupled alternative polyadenylation and splicing (Movassat et al 2016). Additional features contributing to cooperativity may include the local chromatin environment and/or changes in Pol II (Gunderson and Johnson 2009;Gunderson et al 2011;Patrick et al 2015;Milligan et al 2017;Neves et al 2017;Nissen et al 2017). An EM tomography study of the Balbiani ring 3 gene containing 38 introns suggested that only one spliceosome may associate with each nascent RNA at any given time (Wetterberg et al 2001).…”

Section: Discussionmentioning

confidence: 99%

Long-read sequencing of nascent RNA reveals coupling among RNA processing events

2018

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Pre-mRNA splicing is accomplished by the spliceosome, a megadalton complex that assembles de novo on each intron. Because spliceosome assembly and catalysis occur cotranscriptionally, we hypothesized that introns are removed in the order of their transcription in genomes dominated by constitutive splicing. Remarkably little is known about splicing order and the regulatory potential of nascent transcript remodeling by splicing, due to the limitations of existing methods that focus on analysis of mature splicing products (mRNAs) rather than substrates and intermediates. Here, we overcome this obstacle through long-read RNA sequencing of nascent, multi-intron transcripts in the fission yeast Most multi-intron transcripts were fully spliced, consistent with rapid cotranscriptional splicing. However, an unexpectedly high proportion of transcripts were either fully spliced or fully unspliced, suggesting that splicing of any given intron is dependent on the splicing status of other introns in the transcript. Supporting this, mild inhibition of splicing by a temperature-sensitive mutation in, the homolog of vertebrate U2AF65, increased the frequency of fully unspliced transcripts. Importantly, fully unspliced transcripts displayed transcriptional read-through at the polyA site and were degraded cotranscriptionally by the nuclear exosome. Finally, we show that cellular mRNA levels were reduced in genes with a high number of unspliced nascent transcripts during caffeine treatment, showing regulatory significance of cotranscriptional splicing. Therefore, overall splicing of individual nascent transcripts, 3' end formation, and mRNA half-life depend on the splicing status of neighboring introns, suggesting crosstalk among spliceosomes and the polyA cleavage machinery during transcription elongation.

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Section: Discussionmentioning

confidence: 99%

Long-read sequencing of nascent RNA reveals coupling among RNA processing events

2018

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“…Additional features contributing to cooperativity may include the local chromatin environment and/or changes in Pol II elongation and modifications (Gunderson and Johnson 2009;Gunderson et al 2011;Patrick et al 2015;Milligan et al 2017;Neves et al 2017;Nissen et al 2017). An EM tomography study of the Balbiani ring 3 gene containing 38 introns suggested that only one spliceosome may associate with each nascent RNA at any given time (Wetterberg et al 2001).…”

Section: Discussionmentioning

confidence: 99%

Long-read sequencing of nascent RNA reveals coupling among RNA processing events

Herzel

Straube

Neugebauer

2017

Preprint

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Pre-mRNA splicing is accomplished by the spliceosome, a megadalton complex that assembles de novo on each intron. Because spliceosome assembly and catalysis occur co-transcriptionally, we hypothesized that introns are removed in the order of their transcription in genomes dominated by constitutive splicing. Remarkably little is known about splicing order and the regulatory potential of nascent transcript remodeling by splicing, due to the limitations of existing methods that focus on analysis of mature splicing products (mRNAs) rather than substrates and intermediates. Here, we overcome this obstacle through long-read RNA sequencing of nascent, multi-intron transcripts in the fission yeast Schizosaccharomyces pombe. Most multi-intron transcripts were fully spliced, consistent with rapid co-transcriptional splicing. However, an unexpectedly high proportion of transcripts were either fully spliced or fully unspliced, suggesting that splicing of any given intron is dependent on the splicing status of other introns in the transcript. Supporting this, mild inhibition of splicing by a temperature-sensitive mutation in Prp2, the homolog of vertebrate U2AF65, increased the frequency of fully unspliced transcripts. Importantly, fully unspliced transcripts displayed transcriptional read-through at the polyA site and were degraded co-transcriptionally by the nuclear exosome. Finally, we show that cellular mRNA levels were reduced in genes with a high number of unspliced nascent transcripts during caffeine treatment, showing regulatory significance of co-transcriptional splicing. Therefore, overall splicing of individual nascent transcripts, 3’ end formation, and mRNA half-life depend on the splicing status of neighboring introns, suggesting crosstalk among spliceosomes and the polyA cleavage machinery during transcription elongation.

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“…In addition, interference with splicing stimulates pausing at 5 ′ and 3 ′ SSs (Alexander et al 2010b;Chathoth et al 2014). A recent study has shown that the large subunit of Pol II (Rbp1) in yeast is modified by ubiquitination on lysines 452, 695, and 1246 and that Bre5, an RNA binding protein, recruits de-ubiquitinase activity to remove this modifica-tion on K1246 (Milligan et al 2017). A viable deletion of Bre5 or mutation of K1246 to arginine alters Pol II density in intron-containing gene bodies, raising the possibility that de-ubiquitination releases Pol II from splicing-associated pausing and affects the efficiency of splicing.…”

Section: The Coordination Of Splicing With Transcriptionmentioning

confidence: 99%

Nascent RNA and the Coordination of Splicing with Transcription

Neugebauer

2019

Cold Spring Harb Perspect Biol

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At each active protein-encoding gene, nascent RNA is tethered to the DNA axis by elongating RNA polymerase II (Pol II) and is continuously altered by splicing and other processing events during its synthesis. This review discusses the development of three major methods that enable us to track the conversion of precursor messenger RNA (pre-mRNA) to messenger RNA (mRNA) products in vivo: live-cell imaging, metabolic labeling of RNA, and RNA-seq of purified nascent RNA. These approaches are complementary, addressing distinct issues of transcription rates and intron lifetimes alongside spatial information regarding the gene position of Pol II at which spliceosomes act. The findings will be placed in the context of active transcription units, each of which-because of the presence of nascent RNA, Pol II, and features of the chromatin environment-will recruit a potentially gene-specific constellation of RNA binding proteins and processing machineries. Outline 1 Introduction 2 Quantification of splicing relative to transcription using chromatin-based assays 3 Quantification of splicing rates through RNA metabolic labeling 4 Quantification of splicing rates through live-cell fluorescence imaging 5 The coordination of splicing with transcription 6 Concluding remarks References

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RNA polymerase II stalling at pre-mRNA splice sites is enforced by ubiquitination of the catalytic subunit

Cited by 18 publications

References 70 publications

Long-read sequencing of nascent RNA reveals coupling among RNA processing events

Long-read sequencing of nascent RNA reveals coupling among RNA processing events

Long-read sequencing of nascent RNA reveals coupling among RNA processing events

Nascent RNA and the Coordination of Splicing with Transcription

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