Poster abstracts

Poster number 77 submitted by Amelia Cochran

Biochemical and structural analysis of Pus4 mRNA target selection

Amelia Cochran (Chemistry, University of Michigan), Markos Koutmos (Chemistry, University of Michigan), Kristin S. Koutmou (Chemistry, University of Michigan)

Pseudouridine synthase (Pus) enzymes catalyze an isomerization reaction that converts a uridine to a pseudouridine (Ψ), impacting the stability and structure of the modified RNA. Originally characterized as modifiers of tRNA and non-coding RNAs, it is now known that these enzymes also modify mRNA. Pus4, a member of the Trub family, was originally identified as the enzyme responsible for the formation of Ψ55 in most tRNAs. Recent studies have shown that Pus4 is one of the Pus enzymes (along with Pus1 and Pus7) to have many mRNA targets.1 mRNA modifications are likely to impact processing, translation, and degradation of the mRNA molecule, making Pus4 a potential regulator of gene expression. Understanding how Pus4 selects its mRNA targets will be essential in deconvoluting the role of pseudouridine synthases in gene expression regulation. Recent results from a study of Pus7,2 as well as previous characterization of TruB as a tRNA chaperone,3 suggest that Pus enzymes can bind to RNAs with structures and sequences different from their tRNA and ncRNA targets. This has led us to hypothesize that Pus enzymes select their mRNA targets based on consensus sequence, but substrate availability and localization – factors that depend less on Pus4’s ability to selectively bind the substrate, may play a bigger role in target selection. We aim to characterize Pus4’s ability to bind to and modify RNAs of different sequences and structures, and to use crystallography to understand how structural characteristics of Pus4 impact substrate selection.

1.Carlile, T. M.; Rojas-Duran, M. F.; Zinshteyn, B.; Shin, H.; Bartoli, K. M.; Gilbert, W. V. Pseudouridine Profiling Reveals Regulated MRNA Pseudouridylation in Yeast and Human Cells. Nature 2014, 515 (7525), 143–146.
2.Purchal, M. K.; Eyler, D. E.; Tardu, M.; Franco, M. K.; Korn, M. M.; Khan, T.; McNassor, R.; Giles, R.; Lev, K.; Sharma, H.; Monroe, J.; Mallik, L.; Koutmos, M.; Koutmou, K. S. Pseudouridine Synthase 7 Is an Opportunistic Enzyme That Binds and Modifies Substrates with Diverse Sequences and Structures. Proceedings of the National Academy of Sciences 2022, 119 (4), e2109708119.
3.Keffer-Wilkes, L. C.; Veerareddygari, G. R.; Kothe, U. RNA Modification Enzyme TruB Is a TRNA Chaperone. Proceedings of the National Academy of Sciences 2016, 113 (50), 14306–14311.

Keywords: RNA modifications, RNA binding proteins , pseudouridine