Investigations of RNA production and processing in Saccharomyces cerevisiae
Barrass, James David
Over the course of 10 years and 14 publications, 7 of which are described in this thesis, I made significant contributions to the field of RNA processing, particularly RNA splicing in budding yeast. These contributions have been both in knowledge accrued and techniques developed and optimised. For the Ribo1 reporter (chapter 2), I developed the RT-qPCR assays, RNA copy number/cell estimation and 3’ end cleavage assay. These show that the minimum time taken for signal induction, recruitment, assembly of transcription factors and ultimately transcription, is in the order of 4 minutes, with transcription rates of 60 to 90 seconds per kb. The 3’ end cleavage assay reveals that the initial pulse of transcripts may be spliced only partially co-transcriptionally; presumably splicing factors are recruited to the site of transcription during this period. After this initial phase, in the reporter at least, splicing is almost exclusively co-transcriptional. Mutant transcripts on which spliceosomes assemble but are unable to complete splicing, are targeted for very rapid degradation. I have optimised the thio-labelling technique to the point where I can detect thiolated RNAs just 15 seconds after addition of the 4-thiouracil nucleotide analogue to cell cultures (chapter 3). From my data and transcriptome sequencing (chapter 4), I have constructed models of transcription and splicing, which indicate that co-transcriptional splicing is a general feature of most yeast pre-mRNA transcripts and almost all ribosomal protein gene transcripts. Intronic features that act against co-transcriptional splicing include runs of uracils and secondary structure, especially over the branch point adenosine (chapter 4). I have provided a highly detailed protocol and video for thio-labelling of RNA in vivo and its purification (chapter 3). Similarly, I have developed and prepared a comprehensive protocol for auxin-induced protein depletion, to the point where this is the most flexible and least metabolically perturbing technique for doing this in yeast (chapter 6). Applying this method, whilst thio-labelling, I revealed that depleting splicing factors sequesters spliceosome components, rapidly resulting in the cessation of splicing (chapter 7). My microarray results indicate that this is a common consequence of splicing factor deactivation for many or all transcripts. In this thesis I examine RNA metabolism, noting that the processes of transcription, splicing, 3’ end formation and degradation are coordinated and harmonised to optimise fidelity, flexibility and efficiency.