An “AID” to understanding links between splicing and transcription
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Date
29/06/2015Item status
Restricted AccessEmbargo end date
31/12/2100Author
Reid, Jane Elizabeth Anne
Metadata
Abstract
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.