eprintid: 1458117
rev_number: 41
eprint_status: archive
userid: 608
dir: disk0/01/45/81/17
datestamp: 2015-01-30 16:02:09
lastmod: 2019-10-19 07:41:33
status_changed: 2015-01-30 16:02:09
type: thesis
metadata_visibility: show
item_issues_count: 0
creators_name: Robertson, LE
title: In vitro and in vivo studies of RNA recognition by Rna15
ispublished: unpub
divisions: A01
divisions: B02
divisions: C08
divisions: D09
abstract: 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.
date: 2014-12-28
vfaculties: VFLS
oa_status: green
full_text_type: other
thesis_class: doctoral_open
language: eng
thesis_view: UCL_Thesis
primo: open
primo_central: open_green
verified: verified_manual
elements_source: Manually entered
elements_id: 998964
lyricists_name: Robertson, Laura
lyricists_id: LEROB46
full_text_status: public
pagerange: ? - ?
pages: 265
institution: UCL (University College London)
department: Institute of Structural and Molecular Biology
thesis_type: Doctoral
citation:        Robertson, LE;      (2014)    In vitro and in vivo studies of RNA recognition by Rna15.                   Doctoral thesis , UCL (University College London).     Green open access   
 
document_url: https://discovery.ucl.ac.uk/id/eprint/1458117/3/FINAL_CORRECTED_THESIS__SENT__.pdf