Poster abstracts

Poster number 7 submitted by Kira Holton

Substrate selection and functional outcomes by the mRNA-modifying enzyme PUS7

Kira Holton (Department of Biological Chemistry, University of Michigan Medical School), Brittany Bowman (Department of Biological Chemistry, University of Michigan Medical School), Kristin Koutmou (Department of Chemistry, University of Michigan College of LSA), Chase Weidmann (Department of Biological Chemistry, University of Michigan Medical School)

Abstract:
Human RNAs are heavily modified post-transcription, yet the functions of many modifications remain unclear. The enzyme PUS7 is responsible for a large fraction of one such RNA modification, pseudouridylation, where uridine is isomerized to pseudouridine (Ψ). Altered PUS7 activity is implicated in several diseases, but the mechanisms of PUS7-dependent substrate modification and how Ψ contributes to disease pathogenesis are unknown. While PUS7 modifies almost any UNUAR sequence in vitro, only a tiny fraction of these sites are modified inside cells. Without understanding the cellular contexts that direct PUS7 target selection, we cannot predict the functional consequences of PUS7-dependent Ψ in RNA. We hypothesize that distinct RNA structural contexts and protein-RNA interactions drive selection of Ψ sites by PUS7 in cells. We are employing live-cell chemical probing and sequencing technologies to identify these cellular contexts. Protein interaction network probing (RNP-MaP) in human cells finds that Ψ occurs in RNA regions with limited protein binding compared to unmodified UNUAR sites. We are examining whether protein occupancy prevents modification or whether Ψ itself alters protein binding by profiling PUS7-deficient cells. We are similarly probing whether RNA structural motifs are conserved at PUS7-modified sites (by SHAPE-MaP). Additionally, we are developing a luciferase reporter system to measure mRNA expression, processing, and stability in the presence and absence of Ψ. Preliminary results suggest that Ψ-dependent regulation is mRNA-specific. We anticipate that this research will allow us to predict novel PUS7-dependent Ψ sites and may be suggestive of a novel gene regulatory mechanism based on RNA modifications.

Keywords: Pseudouridine, PUS7, RNA-protein interactions