Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.666018
Title: Studies on the metabolism of retained and excised introns in human cells
Author: Hett, Anne
ISNI:       0000 0004 5352 756X
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2014
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
Abstract:
In eukaryotes the coding regions of most genes are interrupted by introns that must be removed by splicing to form a coding mRNA. However, while the splicing mechanism has received a lot of attention, much less is known about the metabolism of introns. This is partly due to the difficulties in studying introns as both aberrantly spliced transcripts and spliced introns are rapidly degraded. In this study, I have analysed intron metabolism in two respects: first I have investigated how introns are degraded following the completion of splicing. Second, I investigate the fate of transcripts, in which introns are retained due to splicing failure. In order to study the degradation of introns following splicing, I performed siRNA mediated knock down of the debrancing enzyme (Dbr1). Following splicing, introns are present in a circular lariat structure and Dbr1 is the enzyme thought to be responsible for opening this. Indeed, I found that knockdown of Dbr1 increased the amount of stabilised introns. Interestingly, introns were found to be stabilised in the cytoplasm and not in the nucleus as expected, even though immunofluoresence showed that Dbr1 is clearly nuclear. However, western blot analysis localised Dbr1 in the cytoplasm. Further investigation showed widely used methods to separate nuclear and cytoplasmic fractions are prone to generating artefacts which result in nucleoplasmic proteins delocalised to the cytoplasm. This finding may prevent future misinterpretation of data obtained by these methods. To investigate splicing failure, it was necessary to generated a sufficiently large pool of unspliced transcripts. To do this I used antisense morpholinos (AMOs) that bind to specific snRNAs (small nuclear RNAs). They are designed to block interaction surfaces that are important for splicing. Using this approach, I investigated the localisation of RNA transcripts and selected RNA processing and degradation factors in normal conditions and where splicing was inhibited. When splicing is inhibited I found splicing factors and unspliced, polyadenylated RNA localising to nuclear, splicing speckle marker SC35 positive speckles. I further discovered that for RNA to localise to nuclear speckles, polyadenylation and RNA cleavage are essential, indicating that SC-35 speckles might sequester unspliced transcripts preventing translation of potentially harmful transcripts. These transcripts remain functional however, and can be spliced where functional spliceosomes can be assembled.
Supervisor: West, Steven; Tollervey, David Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.666018  DOI: Not available
Keywords: introns ; splicing failure ; Dbr1
Share: