Splicing of Nascent RNA Coincides with Intron Exit from RNA Polymerase II

Abstract

Summary Protein-coding genes in eukaryotes are transcribed by RNA polymerase II (Pol II) and introns are removed from pre-mRNA by the spliceosome. Understanding the time lag between Pol II progression and splicing could provide mechanistic insights into the regulation of gene expression. Here we present two single molecule nascent RNA sequencing methods that directly determine the progress of splicing catalysis as a function of Pol II position. Endogenous genes were analyzed on a global scale in budding yeast. We show that splicing is 50% complete when Pol II is only 45nt downstream of introns, with the first spliced products observed as introns emerge from Pol II. Perturbations that slow the rate of spliceosome assembly or speed up the rate of transcription caused splicing delays, showing that regulation of both processes determines in vivo splicing profiles. We propose that matched rates streamline the gene expression pathway, while allowing regulation through kinetic competition.

Extremely fast and incredibly close: cotranscriptional splicing in budding yeast

“…The average position for onset (10% of transcripts spliced) of 26 nt after transcription of the 3 ′ splice site is remarkably soon given that the distance between polymerase and spliceosome active sites is estimated as 24 nt (Carrillo Oesterreich et al 2016). Perhaps this is not surprising: In a first-order kinetics approximation of splicing, the relative time or position of splicing would be an exponential distribution, with a mode at the first available point.…”

“…Do these observations extend to other eukaryotes? For Schizosaccharomyces pombe, fast, cotranscriptional splicing was reported by long-read sequencing (Carrillo Oesterreich et al 2016) and suggested by 4tU-seq (Eser et al 2016). NET-seq in mammals supports cotranscriptional splicing, observing Pol II peaks (interpreted as transcriptional pauses) 3…”

“…1) were a personal communication from K. Harlen, corresponding to Figure 6D of Harlen et al (2016), and we recomputed the fraction spliced using DICE (Huang and Sanguinetti 2016), after aligning raw nascent RNA-seq reads from GEO (GSE68484; SRR2046809) to the S. cerevisiae genome using STAR (Dobin et al 2013). SMIT data are from Carrillo Oesterreich et al (2016), Supplemental Table S1, and 4tU-seq data from Barrass et al (2015), Supplemental Table S7. SMIT intron:exon ratios in Figure 2E were calculated by aligning raw reads from GEO (GSE70908; pooled from smit_20genes and smit_extended data sets, excluding short RNA data) to the S. cerevisiae genome using STAR (Dobin et al 2013); SMIT profiles in Figure 3 and Supplemental Figure S1 were from the same alignments.…”

“…Recently, distinct next-generation sequencing approaches have measured the coupling of transcription to splicing in Saccharomyces cerevisiae: fast metabolic labeling with 4-thio-uracil (4tU-seq) (Barrass et al 2015), nascent RNAseq (Harlen et al 2016), and single molecule intron tracking (SMIT) (Carrillo Oesterreich et al 2016). These approaches produce, for many genes, quantitative estimates of the speed of splicing, extent of cotranscriptional splicing, or polymerase position at splicing, respectively.…”

Circular RNAs: Unexpected outputs of many protein-coding genes

“…This is, in part, because the splicing machinery acts in a co-transcriptional manner to quickly remove most introns as soon as they have been fully transcribed by RNA polymerase II. [18][19][20] By the time a given splice donor (5 0 splice site) is transcribed, all of the upstream splice acceptors (3 0 splice sites) in that pre-mRNA have usually been removed, making backsplicing impossible. However, there are an increasing number of introns that are known to be slowly or post-transcriptionally spliced.…”