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

Abstract

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.

Human co-transcriptional splicing kinetics and coordination revealed by direct nascent RNA sequencing

“…Given that the median transcription rate in both K562 and S2 cells is ~1.25 kb per minute (Ardehali et al, 2009;O'Brien and Lis, 1993;Veloso et al, 2014), our splicing kinetics results are consistent with splicing half-lives estimated from metabolic labeling data: 2 and 14 minutes in Drosophila and mammalian cells, respectively (Pai et al, 2017;Rabani et al, 2014). Our findings suggest that in contrast to S. cerevisiae and S. pombe, in which splicing completes nearly immediately upon synthesis, in metazoan cells Pol II is not in close physical proximity to introns when splicing occurs (Carrillo Oesterreich et al, 2016;Herzel et al, 2018).…”

“…Although enlightening, these strategies fail to uncover the physical link between transcription and splicing, as well as the patterns of splicing across nascent transcripts. Recent work in the yeast species S. cerevisiae and S. pombe suggests that splicing is completed nearly immediately after an intron is synthesized (Carrillo Oesterreich et al, 2016;Herzel et al, 2018), but it remains to be seen whether this is also the case in organisms with more complex gene structures and abundant alternative splicing, such as Drosophila and humans.…”

Aging-associated changes in transcriptional elongation influence metazoan longevity

“…greater splicing efficiency. In the case of co-transcriptional splicing, the removal of lariats depends on the position of Pol-II in the gene (27,28). In this context, our data therefore suggests that accelerated transcription shortens the time to splicing, which reduces the effective half-life of lariats.…”

Revisiting the window of opportunity for co-transcriptional splicing efficiency and fidelity

“…Pre-mRNA splicing is the process that removes the introns and joins the coding segments (exons) to produce mature mRNAs. Evidence accumulating during the last two decades suggests that many, and possibly the majority of, splicing events occur co-transcriptionally, that is before transcription termination [1][2][3][4][5][6][7]. This raises the intruiging possibility of functionally significant interactions between splicing, chromatin, transcription and other RNA processing events, if they occur in close proximity [8][9][10][11][12].…”