Poster abstracts
Poster number 10 submitted by Ben Pockrass
An element in the CFTR 5' UTR represses ribosome recruitment and translation
Ben Pockrass (Department of Biological Chemistry, University of Michigan Medical School), Rachel O. Niederer (Department of Biological Chemistry, University of Michigan Medical School)
Abstract:
The protein output of an mRNA is highly regulated during translation initiation by elements within the 5' untranslated region (5' UTR) of mRNAs, with the identity of the 5' UTR conferring thousand-fold differences in protein output. These elements can form secondary structures, RNA binding protein binding sites, and other features which directly regulate translation initiation. Because translation initiation often determines protein output, 5' UTR regulatory elements can be targeted or engineered to modulate protein output of genes of interest. However, the 5' UTR regulatory landscape for individual genes is largely unknown.
To identify 5' UTR regulatory elements for individual genes, we devised a strategy utilizing Direct Analysis of Ribosome Targeting (DART) to measure ribosome recruitment to a pool of systematically perturbed 5' UTRs. As a proof of principle, we began with the cystic fibrosis transmembrane conductance regulator (CFTR) 5' UTR because it (1) utilizes multiple tissue specific 5' UTR isoforms, (2) contains known regulatory features that can serve as controls, and (3) represents a valuable therapeutic target as the causative gene for cystic fibrosis. To achieve this, we designed a pool of CFTR 5' UTRs containing 6-nucleotide deletions, 6-nucleotide scrambles, and structure probing mutants spanning the entire length of the 5' UTR. We identified a group of deletions within the same 5' UTR region that increased ribosome recruitment compared to the wild type (WT), suggesting this region represses translation initiation in the WT. We used these deletions to narrow down a 7-nucleotide element, which we call a putative repressive element (PRE), within the CFTR 5' UTR that increases ribosome recruitment when deleted. The PRE suppresses translation of a luciferase reporter both in vitro and in vivo, consistent with it acting as a potent repressor. This is not due to differences in mRNA stability, and we are currently performing structure probing and proteomics to explore potential regulatory mechanisms. Using the CFTR 5' UTR as an example, our data demonstrate that DART can successfully identify previously unknown translational control elements that represent promising therapeutic targets.
Keywords: Translation, Initiation, regulation