The W1282X nonsense mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene causes a severe form of cystic fibrosis (CF) which can lead to respiratory failure. However, the treatment for CF caused by the mutation is inadequate. The CFTR-W1282X gene expresses poorly functional, truncated CFTR protein at a very low level, due to nonsense-mediated mRNA decay (NMD). In the context of CF, NMD worsens the clinical outcome of CF patients with CFTR-W1282X mutation by reducing the expression of partially active mutant protein. To develop effective gene-specific therapies for CF, a deeper understanding of the NMD mechanism is needed. NMD requires the deposition of the exon junction complex (EJC) on spliced mRNA. An EJC positioned downstream of a premature-termination codon (PTC) recruits NMD factors, such as SMG6 which mediates endonucleolytic cleavage, a key irreversible step in the EJC-dependent NMD pathway. Our lab showed that multiple downstream EJCs cooperatively potentiate NMD; and synthetic antisense oligonucleotides (ASOs) designed to prevent binding of EJCs downstream of PTCs can attenuate NMD in a gene-specific manner. However, what NMD factors mediate cooperative potentiation of NMD is not understood. CFTR-W1282X lies upstream of three presumptive EJC sites. Whether all three EJCs contribute equally to NMD is unknown. Moreover, whether NMD of CFTR-W1282X mRNA depends on SMG6 has not been investigated. The goal of the proposed research is to elucidate the role of multiple EJCs downstream of the PTC contribute to the NMD of CFTR- W1282X mRNA. Developing ASOs that target each of the downstream EJCs will facilitate the understanding of the mechanism by which individual EJCs potentiate NMD. First, I will develop EJC- targeting ASOs that could restore expression and function of CFTR-W1282X protein in human airway epithelial cells. Second, I will validate the mechanism by which the EJC-targeting ASOs attenuate NMD of CFTR-W1282X mRNA. I will use RNA-immunoprecipitation to compare EJC binding to CFTR mRNA with and without ASO treatment. Third, while SMG6 is the most likely mediator of EJC-dependent NMD, other NMD factors may play partially redundant roles in NMD of CFTR-W1282X mRNA. This may diminish the role of SMG6 in the NMD of CFTR-W1282X in human airway epithelial cells. I will test whether SMG6 plays a major role in EJC-dependent NMD of CFTR-W1282X mRNA, using RNAi knockdown. Also, I will systematically analyze the impact of each downstream EJC on SMG6-mediated endocleavage, by controlling the number and location of downstream EJCs, using both genetic and antisense-directed methods. Results from this project will provide insights into the NMD mechanism, and provide supporting evidence to establish ASO technology as a strategy to restore CFTR function by NMD inhibition.
The W1282X nonsense mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene causes a severe form of cystic fibrosis (CF), one of numerous genetic diseases that could benefit from the gene-targeted suppression of nonsense-mediated mRNA decay (NMD). The goal of the proposed project is to elucidate the mechanism of NMD and develop strategies that restore CFTR gene function by attenuating NMD.
