Post-transcriptional regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by the 5´ untranslated region

Lukowski, Samuel (2010). Post-transcriptional regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by the 5´ untranslated region PhD Thesis, School of Chemistry and Molecular Biosciences, The University of Queensland.

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Author Lukowski, Samuel
Thesis Title Post-transcriptional regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by the 5´ untranslated region
Formatted title
Post-transcriptional regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by the 5´ untranslated region
School, Centre or Institute School of Chemistry and Molecular Biosciences
Institution The University of Queensland
Publication date 2010-08
Thesis type PhD Thesis
Supervisor Ann E. O. Trezise
Joe Rothnagel
Total pages 193
Total colour pages 36
Total black and white pages 157
Language eng
Subjects 0304 Medicinal and Biomolecular Chemistry
Formatted abstract
CFTR is the gene that is responsible for the development of Cystic Fibrosis (CF), a common and fatal disease characterised by chronic and progressive lung disease. The CFTR protein is a chloride channel largely responsible for maintaining cellular fluid homeostasis, but is also involved in complex regulatory interactions with a number of related genes. The most common disease-causing mutation in CFTR, ΔF508, results in protein misfolding and degradation, leading to insufficient chloride excretion from the cell and the disruption of fluid homeostasis. The concept of restoring CFTR channels at the cell surface to relieve the severity of CF disease symptoms has prompted research into the processes that regulate CFTR expression and function. While efforts to understand the mechanisms of CFTR expression and function have mainly focused on transcriptional regulation, post-translational modifications and protein folding, the post-transcriptional processes that dynamically regulate the gene, such as translational efficiency, mRNA stability and RNA protein interactions are not well understood. Post-transcriptional regulation of gene expression is crucial for maintaining normal cellular function. Disruption of these mechanisms can impede protein synthesis, impact upon localisation or promote mRNA degradation, leading to perturbations in cellular pathways and in more extreme cases, disease. Elements present in the 5´ and 3´ untranslated regions (UTR) of the mRNA mediate regulation of gene expression at this level and can influence the protein expression of a large number of mammalian genes.

This thesis focuses on the 5´-untranslated region of the CFTR gene and examines the effects of regulatory elements encoded within upon the translation and stability of the mRNA. Results presented in this thesis suggest that 5´UTR elements such as upstream open reading frames (uORF) and RNA secondary structures contribute to the overall regulation of CFTR expression and that post-transcriptional regulation of CFTR is a significant biological process. In order to understand the molecular mechanisms that regulate CFTR at the post-transcriptional level, an analysis of the CFTR 5´UTR was undertaken to identify functional regulatory motifs using bioinformatics, reporter assays, quantitative real-time PCR and RNA-binding protein assays.

An evolutionarily conserved upstream open reading frame was identified in an array of orthologous vertebrate CFTR sequences, ranging from humans and other mammals through to bony fish. In addition, an RNA hairpin structure, located immediately 3´ of the uORF, was identified in several analysed sequences. The conservation of the uORF and RNA hairpin suggested important regulatory functions that had not been previously characterised in CFTR, but are known to regulate the expression of many other genes. Experiments were conducted to determine the impact of these elements on the regulation of CFTR gene expression, which were found to form a complex, co-operative negative regulatory mechanism that has not been described previously for humans. The components of this mechanism may be suitable targets for small molecules and compounds that can modulate CFTR expression by interacting with these elements.

A CFTR 5´UTR mutation of unknown function was identified by collaborators in a patient presenting with Disseminated Bronchiectasis. Data reported here shows that the CFTR 5´UTR c.-34C>T mutation creates a pathogenic regulatory element that significantly inhibits CFTR translation and reduces mRNA stability, down-regulating expression by 85-99%. This is the first CFTR regulatory mutation (Class V) demonstrated to act at a post transcriptional level and reaffirms the importance of such mutations in multifactorial CFTR-related disease phenotypes. We have shown that the post-transcriptional regulation of CFTR is essential for maintaining normal in vivo gene expression. In addition, this data exemplifies the importance and broad potential of uAUGs and uORFs in the development of human disease.

In the context of both normal cellular physiology and CFTR-related disease development, evidence presented in this thesis suggests that these regulatory elements are directly involved in controlling CFTR gene expression through complex 5´UTR-mediated post-transcriptional mechanisms. Furthermore, these mechanisms may be responsible for the post-transcriptional regulation of many other mammalian genes.

Current literature concerning CFTR gene expression is deficient in the area of post transcriptional regulation. This thesis addresses this deficiency by contributing to the understanding of 5´UTR based regulatory mechanisms in both CFTR and, more generally, in mammalian cells. A novel human regulatory mechanism was characterised and has provided the basis for further experiments that may lead to a specific targeted treatment for CF and CFTR-related diseases. Finally, the characterisation of a uAUG mutation in the CFTR 5´UTR that drastically impedes normal CFTR expression in human cells demonstrates the importance of 5´UTR mutations, their detection and their broader implications in the development of human disease.
Keyword Cystic fibrosis (CF)
CFTR-related disease
Gene expression
Post-transcriptional regulation
Untranslated region

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Created: Mon, 24 Jan 2011, 11:52:56 EST by Mr Samuel Lukowski on behalf of Scholarly Communication and Digitisation Service