This study seeks to address one of the simplest questions that can be asked about an interconnected system; what happens to one process in the absence of the other process? This is a more difficult task than it would appear at first, due to the absence of small molecule inhibitors that can inhibit splicing globally in yeast cells. The first results chapter describes the adaptation of a system called the auxin induced degron (AID) to the task of inhibiting pre-mRNA splicing. This system appears to have several advantages over previous methods of inhibiting splicing and has many potential applications. Another hurdle to understanding what happens to transcription in the absence of splicing is the differential stability of pre-mRNA versus mRNA. At steady state the vast majority of transcripts of a specified gene will be mRNA transcripts. This means that even if you could rapidly inhibit splicing it would be a long time before all the pre-existing mRNA would turn-over. If you waited until specified mRNAs turned over it is likely that the cells would be very sick making it difficult to separate primary and secondary effects. The second results chapter shows the use of a metabolic labelling technique using a uracil analogue called 4-thiouracil (4SU). 4SU is added for an extremely short amount of time (1.5 min, 2.5 min, and 5 min) and the RNA produced during the labelling time is isolated by affinity purification. This allows us to study the kinetics of pre-mRNA splicing in wild-type cells and to seek correlations between splicing kinetics and gene architecture. The third results chapter combines the methods used in the previous two chapters to give a new technique called AID4U-seq. AID4U-seq allows for rapid inhibition of splicing and then the ability to isolate only the transcripts that were created after this inhibition came into effect. This should allow for examination of the primary consequences of blocking pre-mRNA splicing at multiple stages during spliceosome assembly. Additionally AID4U-seq is immediately applicable to the study of other areas of RNA processing. Defining the effects on the transcriptome of inhibiting splicing at multiple stages of assembly is an ambitious aim likely to require many more years of research. Therefore this thesis chiefly seeks to illustrate a novel strategy to begin dissecting a complex issue in which splicing, transcription, degradation and the post-transcriptional modification of histones are all likely to have roles.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:700082 |
| Date | January 2015 |
| Creators | Reid, Jane Elizabeth Anne |
| Contributors | Beggs, Jean ; West, Steven |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/17929 |
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