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Title: Mechanisms of suppression of a pseuoexon in the growth hormone receptor gene
Author: Akker, Scott Alexander
ISNI:       0000 0004 2670 1921
Awarding Body: Queen Mary, University of London
Current Institution: Queen Mary, University of London
Date of Award: 2008
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Pseudoexons are intronic sequences that meet the bioinformatic criteria that define true exons. They outnumber their true counterparts several fold and the reasons for their lack of recognition by the splicing machinery remain unclear. A naturally occurring mutation that activates one such pseudoexon in the growth hormone receptor (GHR) gene has been described. This thesis utilises the mutation as a model of pseudoexon suppression and activation. The aims of the work were to: 1. Establish an in-vitro model of GHR pseudoexon splicing. 2. Identify sequence elements important to the suppression of the pseudoexon. 3. Identify nuclear factors important to the suppression of the pseudoexon. 4. Establish a cell culture system to parallel the in-vitro system. The results show successful establishment of an in vitro and in vivo model of GHR pseudoexon splicing. Deletional experiments using the in vitro splicing system identified silencing elements within the pseudoexon sequence itself. Immunoprecipitation experiments in vitro and RNA interference experiments in vivo implicate heterogenous nuclear ribonucleoprotein El (hnRNP El) as a potential trans-acting silencing factor. Further immunoprecipitation experiments indicate that the spliceosomal factor, Ul small nuclear ribonucleoprotein (Ul snRNP), may also have a role in pseudoexon suppression. Ul snRNP was found to have similar interactions with a panel of other pseudoexons and rarely used alternative exons. We propose a model in which Ul snRNP interacts with silencing factors to block pseudoexon 5' splice-site usage. We suggest that this interaction may occur temporally earlier than true splice site interactions and in a different conformation. Such a temporal and conformationally different interaction would prevent the important Ul snRNA/5' splice-site interaction occurring.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available