Abstract
ABSTRACTThe 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.
Highlights
Splicing involves hundreds of proteins that coordinate the excision of introns from the pre-mRNA, joining the exons and resulting in mature mRNA transcripts
To investigate the splicing of the dystrophin transcript in detail, we performed Illumina HiSeq paired end sequencing on premRNA isolated from 3 differentiated human muscle cell lines
Since dystrophin is expressed at very low levels, we enriched for dystrophin pre-mRNA using a customized library that consisted of biotinylated probes covering all exons, introns, annotated promoters and UTRs of Duchenne Muscular Dystrophy gene (DMD) as well as 3 control genes, excluding repeat masked areas
Summary
Splicing involves hundreds of proteins that coordinate the excision of introns from the pre-mRNA, joining the exons and resulting in mature mRNA transcripts. Multiple alternatively spliced transcripts can be produced from a single transcribed pre-mRNA molecule through a highly regulated process, and its disruption contributes to a large number of human genetic disorders that either directly cause disease or increase disease susceptibility.[1] RNA splicing occurs after assembly of the spliceosome on the pre-mRNA, which includes splice site recognition and intron removal steps.[2] Splice site recognition relies on the identification of exon/intron boundaries This is achieved by 5 (U1, U2/U12, U4/U6 and U5) small nuclear ribonucleoprotein particles (snRNPs), together with more than 100 auxiliary proteins and trans-acting splicing factors (SR proteins and heterogeneous ribonucleoproteins (hnRNPs)).[3,4,5] The correct recognition is supported by cis-acting splicing signals,[6] such as the consensus donor (50) and acceptor (30) splice sites (SS), the branch point sequence (BP) and polypyrimidine tracts (PPT). Additional exonic or intronic splicing enhancers (ESE or ISE) and silencers (ESS or ISS) motifs can influence the inclusion or exclusion of an exon by recruiting trans-acting splicing factors
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