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 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.

Widespread Transcriptional Readthrough Caused by Nab2 Depletion Leads to Chimeric Transcripts with Retained Introns

“…This discovery was facilitated by long-read sequencing of nascent RNA, where entire nascent transcripts are sequenced from the 5 0 end (defined by the transcription start site [TSS]) to the 3 0 end (defined as the position of RNA Pol II at the time of isolation). This method identified a preponderance of ''all or none'' co-transcriptional splicing, where nascent transcripts had all introns spliced and displayed efficient 3 0 end formation or, alternatively, had no introns spliced and displayed readthrough transcription (Herzel et al, 2018). These findings suggest functional coupling between splicing (or retention) of multiple introns and 3 0 end cleavage in vivo.…”

“…Previous work by our lab has identified coordination between co-transcriptional splicing and 3 0 end cleavage in Schizosaccharomyces pombe (Herzel et al, 2018). This discovery was facilitated by long-read sequencing of nascent RNA, where entire nascent transcripts are sequenced from the 5 0 end (defined by the transcription start site [TSS]) to the 3 0 end (defined as the position of RNA Pol II at the time of isolation).…”

RNA Polymerase II CTD phosphatase Rtr1 fine-tunes transcription termination

“…The termination of transcription by eukaryotic RNA Polymerase II (RNAPII) is tightly coupled with RNA processing, including small RNA processing, splicing, and mRNA cleavage and polyadenylation at the 3'-end of protein-coding genes [1,2]. Recent studies have reaffirmed that transcription termination in eukaryotes is a highly dynamic process that can lead to different gene expression outputs through mechanisms, such as alternative polyadenylation site usage and premature transcription termination [3][4][5][6][7][8][9][10].…”

Calculating the Most Likely Intron Splicing Orders in S. pombe, Fruit Fly, Arabidopsis thaliana, and Humans

“…One natural line of thought is that since splicing is coupled with transcription, the splicing order may also be consistent with the transcriptional direction [6].…”

“…However, several recent studies have shown that this is not the case; these studies used long-read sequencing or bulk short-read sequencing to find that splicing is a co-transcriptional process but is not always consistent with the direction of transcription in S. pombe, fruit fly and humans [6][7][8]. In addition, intron splicing order has been shown to influence alternative splicing in COL5A1 [9], and the importance of intron splicing order has also been indicated by two recent studies that proved that intron splicing order can affect splicing fidelity [10,11].…”