Talk abstracts
Talk on Friday 01:45-02:00pm submitted by Audrey Hoelscher
Systematic analysis of ribosome recruitment to trinucleotide repeats
Audrey Hoelscher (University of Michigan, Department of Biological Chemistry), Ben Pockrass (University of Michigan, Department of Biological Chemistry), Jacob Horn (University of Michigan, Department of Biological Chemistry), Leidy Vanegas Cano (University of Michigan, Department of Biological Chemistry), Sinem Ovunc, Peter Todd (University of Michigan, Department of Neurology), Rachel Niederer (University of Michigan, Department of Biological Chemistry)
Abstract:
Trinucleotide repeat expansions are associated with a number of neurological disorders such as Huntington’s disease, spinocerebellar ataxia, and frontotemporal dementia. Multiple mechanisms can underlie these diseases, including Repeat Associated non-AUG (RAN) translation. RAN translation results in aberrant and aggregation-prone protein products, however the mechanisms used by the repeat elements to recruit translation machinery remain unclear. To investigate the molecular requirements for RAN translation, we used Direct Analysis of Ribosome Targeting (DART) to systematically test ribosome recruitment to UTRs with various trinucleotide repeats. We tested multiple aspects of the repeats by varying the repeat nucleotide identity, position within the 5’ UTR, and length. To investigate ribosome recruitment in a relevant context, we performed this DART assay in rat cortical lysates. Overall, we measured ribosome recruitment levels for 24,000 different sequences which revealed both expected and unexpected trends within the data. Structured elements positioned near the mRNA cap were repressive to translation as expected. Interestingly, long stretches of AAA repeats were repressive regardless of position. Finally, because RAN translation often proceeds via cap-independent mechanisms, we also performed DART on an uncapped version of the pool. Surprisingly, we found a single unique group of transcripts which showed high uncapped ribosome recruitment while capped ribosome recruitment remained low. All transcripts in this subgroup contained a version of the GUC trinucleotide, typically 20 repeats in length and positioned closer to the start codon, indicating that this sequence may be allowing initiation to occur more efficiently than expected for uncapped mRNA. These results suggest a novel context where cap-independent initiation may occur that could be relevant to our understanding of repeat expansion disease and translation regulation.
Keywords: trinucleotide repeats, translational control, 5UTR