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Title: The regulation of a novel exon in the rat α-tropomyosin gene
Author: Coles, J. L.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2007
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A conserved novel exon has been identified downstream of exon 3 of the rat α-tropomyosin gene. Inclusion of this exon is predicted to result in nonsense mediated decay of the α-tropomyosin transcript due to the presence of premature termination codons. Previous work showed that this nonsense exon is readily activated by a number of mutations in the 3’splice site and the 5’ end of the exon. Furthermore, there is evidence that the nonsense exon is activated in kidney, heart and lung tissue, indicating that its inclusion may be regulated in a tissue-specific manner. In this study, several computational data-sets were used to predict the locations of auxiliary cis-acting regulatory elements along the length of the nonsense exon. This information was used to design mutants in the putative regulatory elements, which revealed the presence of at least five exons splicing silencers (ESSs) and four exon splicing enhancers (ESEs). A number of trans-acting factors were subsequently identified as regulating the inclusion of the nonsense exon. These include hnRNP H and hnRNP F, the binding of which to a G-rich ESS at the 5’ end of the nonsense exon is required for repression. hnRNP A1 and CELF4 activate nonsense exon inclusion possibly by acting as anti-repressors of hnRNP H and hnRNP F. In addition SRp30c was identified as an activator of the nonsense exon, whilst SRp20 and hnRNP I (PTB) were identified as additional repressors, though the binding sites for SRp30c and SRp20 have yet to be elucidated. Inclusion of the nonsense exon was shown to result in NMD of the transcript in an α-tropomyosin-based reporter construct.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available