Talk abstracts
Talk on Friday 03:30-03:45pm submitted by Kira Holton
Substrate Selection and Functional Outcomes by the mRNA modifying enzyme PUS7
Kira Holton (Department of Biological Chemistry, University of Michigan), Brittany Bowman (Department of Biological Chemistry, University of Michigan), Kristin Koutmou (Department of Chemistry, University of Michigan), Chase Weidmann (Department of Biological Chemistry, University of Michigan)
Abstract:
Human RNAs undergo a variety of post-transcriptional modifications, and the extent and effect of most of these modifications is currently unknown. The enzyme PUS7 is responsible for a large fraction of one such RNA modification, pseudouridylation, where uridine bases are 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 unclear. While PUS7 can modify almost any UNUAR sequence when reconstituted in solution, only a tiny fraction of these sites (< 3 %) are modified inside the cell. 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 modification occurs in RNA regions with limited protein binding, when compared to unmodified UNUAR sites. We are profiling protein engagement at these sites in cells lacking PUS7 to determine whether protein occupancy inhibits Ψ modification or alternatively if Ψ modification limits protein binding. We are similarly probing whether RNA structural motifs are conserved at PUS7-modified sites (by SHAPE-MaP). Additionally, we are concurrently developing a cellular luciferase-based reporter assay to measure mRNA expression, processing, and stability in the presence and absence of Ψ. Preliminary data suggests that Ψ-dependent regulation is mRNA-specific. We anticipate that the knowledge generated from 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: RNA Modification, PUS7, RNA-Protein Interactions