Fig. 8

PRPF8 depletion alters the dynamics of RNA splicing during transcription. a, b Intronic reads contain potential information on the dynamics of splicing. Introns located towards the 5′ end of transcripts have a higher probability of being spliced out under a scenario of co-transcriptional splicing, but this is not the case if splicing occurs post-transcriptionally. A co-transcriptional splicing ratio was calculated by comparing the intronic coverage of the first versus last introns in each transcript, as indicated in (b), with the values used to calculate the ratio shown schematically in (a). Such coverage was normalized to take into account intron length and the expression levels of adjacent exons. A representative example is shown in (a), with first and last intronic reads indicated by arrowheads. RPKM Reads per kilobase of exon per million reads mapped. c Distribution of the co-transcriptional splicing ratio in control siRNA-treated and PRPF8-depleted samples. Co-transcriptional splicing dominates in control siRNA-treated samples as indicated by the low ratio values. The co-transcriptional splicing ratio significantly increases following PRPF8 depletion, suggesting disruptions in the co-transcriptional splicing efficiency (p < 5.34 × 10⁻⁹). Only genes with one transcript annotated in Ensembl 66 were considered for this analysis (n = 2366; “Materials and methods”). d PRPF8 depletion alters the dynamics of RNA splicing during transcription. The kinetics of transcription and splicing recovery of CDC20, APC8, and ASPM genes following release from drug-induced transcriptional arrest were measured in control siRNA-treated and PRPF8-depleted cells using a previously published protocol [37]. Primer pairs used are indicated and were chosen to measure the dynamics of RNA splicing of introns that are either retained or exons that are differentially used as determined by RNA sequencing experiments. As a control, the kinetics of splicing of an exon that was not differentially used in the ASPM transcript was also measured. PRPF8 depletion inhibits the kinetics of splicing as the delay between detection of the unspliced nascent transcript (indicated in black) and the partially spliced transcript (indicated in light gray) is ~20 minutes for CDC23 and ~15 minutes for ASPM in control siRNA-treated cells, but in excess of 40 minutes in PRPF8-depleted cells. In contrast, the kinetics of splicing and removal of intron 1 in the ASPM transcript is similar in both conditions. mRNA expression levels were normalized to non-DRB-treated cells for each condition (control siRNA-treated and PRPF8-depleted cells), which were harvested alongside the cells just released from DRB (0 minute time-point). DRB 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside