Pre-mRNA splicing is a critical and regulated processing event where introns are precisely excised from nascent RNA transcripts. As many as one third of all heritable disease mutations result in splicing defects. To better understand the mechanism of splicing we propose a large-scale implementation of classic in vitro splicing assays and assembly assays. We propose to use this technology to map splicing elements around the 3'ss, to map branchpoints upstream of the 3'ss and also to repurpose spent exome sequencing libraries as in-vitro splicing substrates. This final goal stages an in-vitro splicing assay on characterized, genotyped samples with the aim of extracting phenotypic data from a genotyping tool. The existence of millions of characterized sequencing libraries could potentially connect in-vitro discovery to clinically relevant patient diagnosis.
A great deal of research effort has been invested in understanding how genes are expressed. In vivo profiling experiments have recorded which transcripts accumulate in cells across different tissues. As part of the technological revolution of the post genomic era, these measurements are made at a grand scale (i.e. thousands of transcripts followed simultaneously). However the mechanisms of splicing have been determined for only a few model substrates. Here, we remedy this deficiency by implementing in-vitro mechanistic studies at a genomic scale. We propose to map functional elements and rare branched intermediates for all the introns in the genome. These in vitro recordings will provide a vital piece of information to complement in vivo studies and help us better understand biology and disease.
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