The kinetics of pre-mRNA splicing in theDrosophilagenome: influence of gene architecture

Abstract

Production of most eukaryotic mRNAs requires splicing of introns from pre-mRNA. The splicing reaction requires definition of splice sites, which are initially recognized in either intron-spanning ("intron definition") or exon-spanning ("exon definition") pairs. To understand how exon and intron length and splice site recognition mode impact splicing, we measured splicing rates genome-wide in Drosophila, using metabolic labeling/RNA sequencing and new mathematical models to estimate rates. We found that the modal intron length range of 60-70 nt represents a local maximum of splicing rates, but that much longer exon-defined introns are spliced even faster and more accurately. Surprisingly, we observed low variation in splicing rates across introns in the same gene, suggesting the presence of gene-level influences, and we identified multiple gene level variables associated with splicing rate. Together our data suggest that developmental and stress response genes may have preferentially evolved exon definition in order to enhance rates of splicing.

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

“…RNA purified from the chromatin fraction is then subjected to 4sU pu-rification. Even for much longer periods than 8 min, 4sU treatment does not show a decrease in splicing levels (Schofield et al, 2018) (Figures S1B and S1C), supporting the use of this metabolic labeling approach to quantify splicing rates (Pai et al, 2017;Rabani et al, 2014;Wachutka et al, 2019). The combination of cellular fractionation and 4sU purification enriches for RNAs that are both recently transcribed and localized to chromatin.…”

“…A variety of approaches have provided estimates of the length of time between the synthesis of an intron and its removal by splicing in living cells. Measurements of intron synthesis, lariat degradation, and the completion of the splicing reaction by quantitative PCR (Singh and Padgett, 2009), metabolic labeling (Pai et al, 2017;Rabani et al, 2014;Wachutka et al, 2019;Windhager et al, 2012), or single-molecule imaging (Coulon et al, 2014;Martin et al, 2013) have revealed that intron removal can take between 30 s and 1 h in mammalian cells. Although enlightening, these strategies fail to uncover the physical link between transcription and splicing, as well as the patterns of splicing across nascent transcripts.…”

Spatial organization of transcript elongation and splicing kinetics

“…Previous studies have shown that the splice sites of adjacent introns can act synergistically to more effectively recruit spliceosomes 22,23 , which could also facilitate the stabilization of rates within genes. Metabolic labeling studies in D. melanogaster showed that introns within the same gene have consistent half-lives 12 . Analysis of our measured rates shows that co-transcriptional splicing kinetics are highly coordinated within gene bodies in human cells (Fig.…”

Transcription and splicing dynamics during early Drosophila development

“…Intriguingly, however, for very large introns (>1000 nt), the proportion with SR > 0 increases again, suggesting that specific mechanisms may have evolved for the intron-defined splicing of very large introns. The tendency for faster splicing kinetics with average intron sizes has also been reported using metabolic labelling in both Drosophila (Pai et al 2017) and human cells (Windhager et al 2012).…”

“…In addition, Pai et al (2017) found that GC poorer introns were spliced faster. We uncovered a similar relationship with GC content, with the highest proportion of introns with SR > 0 observed when both the exon and the intron were AT-rich but the exon had a higher GC content than the intron.…”