Poster abstracts
Poster number 128 submitted by David Muzyka
Quantitative analysis of PUS enzymatic activity via Triple Quad Mass Spectrometry
David Muzyka (Department of Chemistry, University of Michigan), Amelia Cochran (Department of Chemistry, University of Michigan), Lauren Barnes (Department of Chemistry, University of Michigan), Kristin Koutmou (Department of Chemistry, University of Michigan)
Abstract:
Pseudouridine Synthases (PUS) enzymes are responsible for the isomerization of uridine to pseudouridine (Ψ). Ψ is the most abundant chemical modification found across all types of RNA, and because it is chemically very similar to uridine, this presents challenges for its rigorous, quantitative detection. We developed an assay to measure the degree of pseudouridylation of RNAs by digestion into nucleosides followed by analysis via LC-MS/MS. This assay was validated by measuring the inclusion of Ψ by the enzyme PUS7, a eukaryotic PUS enzyme that modifies both non-coding (i.e. tRNA) and coding (mRNA) RNAs at UNUAR sequence motifs in cells. With this method in hand, we have begun exploring how other PUS enzymes select their substrates. Our initial studies focused on comparing the activities of homologous pseudouridine synthase enzymes, PUS4 and TruB from S. cerevisiae and E. coli, respectively. Despite sharing about 30% sequence homology, PUS4 has a much larger reported substrate scope in vivo than TruB. However, whether this is due to changes in inherent enzyme activity, or enzyme regulation (or both) remains to be discovered. Our initial data reveal that purified PUS4 and TruB can both modify an array of substrates in vitro, though PUS4 modifies some non-tRNA more efficiently.
References:
Purchal et al., 2022
Keywords: RNA Modifications, Mass Spectrometry