For successful gene expression in eukaryotes, mRNA transcripts are processed in the 5’ and 3’ untranslated regions (UTRs) and non-coding mRNA is spliced out. These processes are crucial in determining the fate of the mRNA transcript. Rna14 and Rna15 are subunits of Cleavage factor 1A (CF1A) and required for 3’ end processing in S. cerevisiae. Structural and biophysical data have determined a number of residues within the RNA recognition motif (RRM) of Rna15 that interact directly with RNA. However, although the crystal structure of the RRM revealed a mechanism for the preferential recognition of G/U nucleotides by Rna15 the sequence specificity for Rna15 is still extensively debated. This thesis applies combination of in vivo and in vitro techniques aimed to characterise Rna15-RNA binding, Rna15 interaction with Rna14 and examine consequences for RNA processing and yeast viability in vivo. An in vitro mutational/biophysical analysis is presented that reveals the residues essential for the Rna15-RNA interaction and application of NMR-quantified Scaffold Independent Analysis (SIA) demonstrates a clear GU-bias in the in vitro consensus sequence. However, given these strong effects, surprisingly only extensive mutation of the RRM produces growth defects in S. cerevisiae and qRT-PCR experiments employing a small subset of genes show only slight effects on polyA site selection. By contrast, an RNA-sequencing (RNA-Seq) global analysis of expression and transcriptional readthrough reveals that expression of over 100 S. cerevisiae genes is severely affected when the RRM of Rna15 is deleted and in addition the 3’-UTR of the mRNA of a sample set of 40 genes is significantly different to wild type. These results indicate that only severe reduction of Rna15-RNA interactions result in defects in transcriptional and 3’ end processing, hypothesized to be due in part to functional redundancy. Nevertheless, the global changes observed upon deletion of the Rna15 RRM are striking and reinforce the link between 3’-end processing, transcriptional regulation and gene expression.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:634701 |
| Date | January 2014 |
| Creators | Robertson, L. E. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1458117/ |
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