Poster abstracts
Poster number 162 submitted by Alice Youle
Translation of CFTR Proceeds from Multiple Start Sites
Alice Youle (Biological Chemistry, University of Michigan), Ben Pockrass (Biological Chemistry, University of Michigan), Rachel O. Niederer (Biological Chemistry, University of Michigan)
Abstract:
Cystic fibrosis is a monogenic disorder primarily caused by mutations to the chloride ion channel protein CFTR. Over 2000 causative genomic variants have been identified, which result in a wide range of molecular phenotypes, including low expression of CFTR, or expression of CFTR with reduced or abolished function. The amount of functional CFTR is tightly connected to the severity of disease, which has focused therapeutic efforts on increasing expression of functional CFTR. The 5′UTR of the CFTR mRNA is highly structured and contains one upstream open reading frame, suggesting it is a regulatory hub for controlling CFTR levels. However, the functional impact of these RNA features has not been directly tested. Here, we sought to fully characterize the regulatory features within the CFTR 5′UTR with the goal of identifying potential therapeutic targets. We designed systematic mutations across the entire 5′UTR including deletions, sequence scrambles and structure disruptions and measured ribosome recruitment in high throughput using Direct Analysis of Ribosome Targeting (DART). Surprisingly, we find that the uAUG in the CFTR 5′UTR is not inhibitory for translation of CFTR as expected, but rather enhances translational output. Further, we show that translation initiation occurs on at least one noncanonical upstream start site in addition to the annotated uAUG. These findings reveal unexpected modes of regulation within the CFTR 5′UTR, uncovering novel therapeutic targets for cystic fibrosis patients and establishing the CFTR 5′UTR as a model for determining the mechanistic details of translation start site fidelity.
Keywords: 5UTR, cystic fibrosis, start site selection