Poster abstracts

Poster number 29 submitted by Amelia Cochran

Investigation of Pus4 Substrate Selection

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

Abstract:
Pseudouridine synthase (Pus) enzymes catalyze the isomerization of uridine to 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 enzyme 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 also target mRNA sequences. mRNA pseudouridine modifications have been shown 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 its role (and the role of pseudouridine synthases) in gene expression regulation. Recent characterizations of Pus7 and Pus4 show that some Pus enzymes can bind to and modify RNAs with structures and sequences different from their tRNA and ncRNA targets. This points to the possibility that Pus enzymes target RNAs with less specificity than previously thought. These observations lead us to hypothesize that while the substrate scope of Pus enzymes is loosely guided by mRNA substrate properties (such as consensus sequence and/or structure), substrate availability and localization – factors that depend less on the enzyme’s ability to selectively bind the substrate, may play a bigger role in target selection. In order to further understand Pus4 specificity, we are working to characterize Pus4’s ability to bind to and modify RNAs of different sequences and structures, and we are using crystallography to understand how structural characteristics of Pus4 impact substrate selection. Our initial findings suggest that there may be subtle differences in how Pus enzymes select their substrates.

Keywords: RNA modifications, RNA binding proteins , pseudouridine