Talk abstracts
Talk on Friday 03:45-04:00pm submitted by Alice Youle
High-throughput discovery of 5UTR structures that regulate translation
Alice Youle (Biological Chemistry, University of Michigan), Rachel O. Niederer (Biological Chemistry, University of Michigan)
Abstract:
RNA secondary structures have functions in all RNA types across all domains of life. Well-characterized examples include tRNAs and riboswitches, where their specific structure is absolutely critical for mediating their respective functions. In contrast, functional structures within mRNAs are comparatively hard to find and consequently less well characterized.One major challenge is that structures within mRNAs do not share a unifying function, which makes it challenging to disentangle which specific structures are important versus those that are simply thermodynamically favorable. Here, we have combined SHAPE-MaP (1) and high-throughput compensatory mutations with Direct Analysis of Ribosome Targeting (DART) (2) to discover novel mRNA secondary structures impacting translation initiation. We selected 12,000 human 5UTRs from disease-relevant genes, whose folded 5UTRs range in predicted minimum free energy from -100 to -1 kcal/mol. For 400 sequences, we introduced structure-disrupting and compensatory mutations to directly test the role of the predicted secondary structure in translation initiation. We then measured folding of the entire pool in high-throughput using SHAPE-MaP. Lastly, we performed DART to measure ribosome recruitment to each 5UTR. Importantly, this pipeline enables us to identify specific structures impacting translation as opposed to base pairing exerting a more generic effect. Secondary structure has primarily been associated with translation inhibition, so we were surprised to discover almost an equivalent number of stimulatory and repressive structures in this pool. Of note, the stimulatory 5UTR structures are associated with genes whose 5UTRs are largely uncharacterized and are involved in a wide range of diseases. This approach offers a powerful strategy to systematically identify and functionally characterize secondary structures that regulate translation, and may uncover potentially novel avenues for therapeutics.
References:
(1) Siegfried NA, Busan S, Rice GM, Nelson JA, Weeks KM. RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP). Nat Methods. 2014
(2) Niederer RO, Rojas-Duran MF, Zinshteyn B, Gilbert WV. Direct analysis of ribosome targeting illuminates thousand-fold regulation of translation initiation. Cell Systems. 2022
Keywords: Secondary Structure, 5UTR, Translation