Development of new chemical biological tools to probe splice site selection
thesisposted on 13.03.2013, 13:45 by Helen Lewis
RNA splicing is a key process in gene expression and regulation in Eukaryotes and involves the processing of pre-mRNA sequences into mature mRNA. Pre-mRNA consists of exons (protein coding regions) and introns (non-protein coding regions). The introns of the pre-mRNA are excised and the exons ligate to form mature mRNA ready for export from the nucleus. Within the pre-mRNA there are numerous splice sites, some of which are conserved whilst others are alternative splice sites. RNA splicing can follow two different pathways: constitutive or alternative and in humans around 90% of pre mRNA is alternatively spliced which accounts for the formation of multiple isoforms of a single gene. The regulation of splicing involves cis-acting factors which are enhancer/silencer sequences within the pre-mRNA and trans-acting factors comprising of cellular factors including RNA and proteins, combined together they enhance or silence splicing. A major challenge in the field is to determine the interplay between the various factors associated with the promotion or silencing of specific splice sites. Two putative models for the utilization of splice sites have been proposed; firstly, a looping mechanism whereby the enhancers randomly collide with each other by 3D diffusion forming a loop. Secondly, enhancer mediated splicing occurs by a cooperative protein binding process. However, due to the limitations of current biochemical tools, it is not possible to determine the exact mode of action. In this thesis, a new chemical biological approach has been developed which addresses this question, involving the construction of tripartite RNA constructs separated by a non-RNA tether. Using these model systems, compelling evidence is provided which demonstrates splice site selection does not proceed via a looping mechanism which is the widely accepted model in the field.