Genome-wide analysis for the identification of mRNA elements that contribute to post-transcriptional regulation of gene expression

Post-transcriptional regulation is an important step in the flow of genetic information from genome to proteome. It has been shown to play vital roles in normal cellular processes; embryonic development; and many pathological states such as cancer, cardiovascular disorders and obesity. Despite its importance, much remains to be learned about the molecular mechanisms of post-transcriptional control. The work described in this thesis employs the use of genome-wide study to identify mRNA features that mediate ribosome recruitment and mRNA decay in immortalized cell lines and primary cell lines and develops an approach to systematically identify mRNA regulatory elements in subsets of transcripts by comparison and integration of current computational methods.;In the study of a model system in which apoptosis resistance was conferred upon NIH3T3 cells by overexpression of eIF4E, we showed that there was a set of transcripts that manifested an increase in translational efficiency during eIF4E-mediated escape from apoptosis. Using conventional motif discovery tools, we identified a novel 55nt RNA consensus hairpin structure that was conserved in the 5' untranslated region of translationally activated transcripts. This structure was sufficient to target a reporter mRNA for translational activation under pro-apoptotic stress in cells overexpressing eIF4E, but not in wild type cells.;In another example, an RNA element mediating mRNA decay was explored in primary human T cells. We used an enumeration and clustering method to identify a consensus GU -- rich element (GRE) that was enriched in short-lived transcripts. Insertion of GRE-containing sequences from c-jun, jun B, or TNF receptor 1B, but not mutated GRE sequences, into the 3'UTR of a beta-globin transcript conferred instability on the otherwise stable beta-globin transcript. CUG-binding protein 1 (CUGBP1) was identified as the GRE binding protein. This work established that GRE and CUGBP1 are regulators of a novel mRNA decay pathway.;To develop an approach to efficiently identify putative regulatory elements from sets of co-regulated genes, we compared the performance of current analytical methods for element discovery based on three different principles: enumeration, optimization and the principle based on sequence/structure conservation. Three types of elements were examined, elements conserved in both primary sequence, and secondary structure, elements conserved only in primary sequence and microRNA targets. Our results indicated that each method has advantages when used to identify a spectrum of RNA regulatory elements; and that integrating the output from all 3 algorithms leads to the most complete identification of elements. To facilitate future laboratory-based studies of co-regulation mechanisms, we developed an approach presented as a web service (https://dbw.msi.umn.edu:8443/recit) that can be used to identify the most promising elements from genome-wide post-transcriptional profiling data sets.;Using this approach in a genome-wide study of idiopathic pulmonary fibrosis (IPF), a prototype fibrotic disease, we showed that pathological translational control was a major characteristic of IPF myofibroblasts; and that these elements provided potential mechanisms of translational co-regulation in IPF. We also found systems-level indications for TGFbeta-driven epithelial to mesenchymal transition (EMT) as one source of IPF myofibroblasts.;Together these findings provide important mechanistic insights into post-transcriptional control in normal biology and disease.