Poster abstracts
Poster number 92 submitted by Cesar Martinez
Precise mapping of translation initiation sites using the bacterial toxin RelE
Cesar Martinez (University of Michigan, Chemical Biology), Rachel Niederer (University of Michigan, Chemical Biology), Ross Kaufhold (University of Michigan, Chemical Biology)
Abstract:
Modulation of gene expression occurs at multiple levels such as the transcriptional, translational, and post-translational stages. Translational control of gene expression is of interest due to its pervasiveness in various biological processes from early development to diseases such as cancer. High throughput assays that measure translational output have been developed to decipher the role of the 5’ untranslated region (5’ UTR) of mRNA in translational control of gene expression. Currently, these assays lack the resolution to pinpoint the exact ribosome binding site during translation initiation. This information would be especially useful in contexts where translation goes awry such as mRNAs with multiple start codons or RAN (repeat-associated non-AUG) translation. To achieve our aim of increasing their resolution, we set out to incorporate the bacterial toxin, RelE, into high-throughput translation assays to precisely map the ribosome’s position during translation initiation. RelE cleaves ribosome-bound mRNA in the ribosomal A site, enabling us to identify the site of initiation. We generated three RNAs to use as benchmarks for our experiment that contain one or two start codons or a short stem loop. We then performed an in vitro translation assay to load ribosomes onto these mRNAs and used RelE to cleave the transcripts at the position where the ribosome was bound. Addition of RelE dramatically reduces protein output from the in vitro translation assay, indicating that the protein is active and likely cleaving RNA. Next, we will determine the sites of cleavage on the RNAs by primer extension. In the future, this method can be incorporated into previously developed high-throughput translational assays to assign positional ribosome recruitment scores. Using RelE to further explore the process of translation initiation in RAN translation could potentially reveal how certain neurodegenerative diseases manifest in the human body and may identify potential treatments.
Keywords: translation initiation, bacterial toxin RelE, 5 UTR