Non-sequential and multi-step splicing of the dystrophin transcript

Abstract

The dystrophin protein encoding DMD gene is the longest human gene. The 2.2 Mb long human dystrophin transcript takes 16 hours to be transcribed and is co-transcriptionally spliced. It contains long introns (24 over 10kb long, 5 over 100kb long) and the heterogeneity in intron size makes it an ideal transcript to study different aspects of the human splicing process. Splicing is a complex process and much is unknown regarding the splicing of long introns in human genes.Here, we used ultra-deep transcript sequencing to characterize splicing of the dystrophin transcripts in 3 different human skeletal muscle cell lines, and explored the order of intron removal and multi-step splicing. Coverage and read pair analyses showed that around 40% of the introns were not always removed sequentially. Additionally, for the first time, we report that non-consecutive intron removal resulted in 3 or more joined exons which are flanked by unspliced introns and we defined these joined exons as an exon block. Lastly, computational and experimental data revealed that, for the majority of dystrophin introns, multistep splicing events are used to splice out a single intron.Overall, our data show for the first time in a human transcript, that multi-step intron removal is a general feature of mRNA splicing.

Endogenous Multiple Exon Skipping and Back-Splicing at the DMD Mutation Hotspot

“…Indeed, they characterized DMD introns into non-sequential introns (including post-transcriptionally spliced introns:post-transcriptional introns) and sequential introns (so-called co-transcriptionally spliced introns or co-transcriptional introns). DMD intron 44 was annotated as a post-transcriptional intron [28]; therefore, it was reasonable to suggest that it could take part in intronic interactions with downstream introns for the MES events and the generation of circRNA, similar to the current model described above. This is the first report to suggest a relationship between MES with circRNA generation and post-transcriptional introns.…”

“…Recently, Gazzoli et al reported non-sequentially spliced introns and sequentially spliced introns of the DMD gene by capturing pre-mRNA sequencing [28]. A sequentially spliced intron could be considered as a co-transcriptionally spliced intron (co-transcriptional intron).…”

“…A sequentially spliced intron could be considered as a co-transcriptionally spliced intron (co-transcriptional intron). Non-sequentially spliced introns included not only post-transcriptionally spliced introns, but also introns involved in recursive splicing and nested splicing [28,29,30]. Recursive and nested splicing produce intermediate pre-mRNAs by intra-intronic splicing.…”

“…Only the upstream adjacent intron to exon 58 was a post-transcriptional intron, and the others were co-transcriptional introns [28]. Therefore, we focused on active sites for back-splicing.…”

“…We also observed that the 3′ end of exon 44 was more efficiently involved not only in the generation of exon 44–56, but also in other MESs around the DMD hotspot. Recently, Gazzoli et al reported an initial view of post-transcriptional DMD pre-mRNA in muscular cells [28]. Indeed, they characterized DMD introns into non-sequential introns (including post-transcriptionally spliced introns:post-transcriptional introns) and sequential introns (so-called co-transcriptionally spliced introns or co-transcriptional introns).…”