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|Title:||The influence of pre-mRNA primary and secondary structure in splicing.|
|Authors:||Turnbull-Ross, Andrew D.|
|Presented at:||University of Leicester|
|Abstract:||This thesis describes work undertaken on two aspects of eukaryotic pre-messenger RNA splicing. The first study investigated the use of oligonucleotide-limited transcription to produce model substrates for in vitro splicing reactions. It was found that the length of 3' terminal exon of the pre-mRNA could be reduced to four nucleotides, and possibly less, and both steps of splicing could still be observed. However at short 3'exon lengths, step 2 (exon ligation and release of lariat intron) was inefficient. This data correlated with previous studies of splicing complex formation on truncated pre-mRNAs and, in addition, suggested transient non-specific binding between the 3'exon and the splicing complex was required to activate the 3'splice site during step 2. The effect of changing the 3'exon sequence to either 5'- or 3'splice site sequences was also investigated. Mutation to a 5'splice site sequence had no effect on splicing efficiency at the authentic splice sites, while mutation to a 3'splice site resulted in enhanced 3'- 5'exonuclease degradation of the lariat-3'exon splicing intermediate.;The second study tested a number of models explaining the mechanism of developmental1y regulated alternative 3' end processing of murine immunoglobulin M pre-mRNA. The system contains two polyadenylation sites one of which is located within the 3' terminal intron. Competition has been proposed to occur between the two polyadenylation sites or between splicing of the terminal intron and polyadenylation at the site within the intron. The data produced suggest the competition is principally between the splicing reaction and use of the polyadenylation site within the intron. It was also found that the intron 3'splice site was sub-optimal due to a small stem-loop structure which sequesters the polypyrimidine tract and cleavage site. This is the first demonstration of a naturally occuring secondary structure modifying the in vivo efficiency of a splice site.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Biochemistry|
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