A Shared Structural Recognition Element in mRNA Substrates of the tRNA Modifying Enzyme Pseudouridine Synthase 1

• Published in: The FASEB journal Vol. 31; no. S1
• Main Authors: Carlile, Thomas M, Bell, Tristan A., Rojas‐Duran, Maria F, Zinshteyn, Boris, Shin, Hakyung, Mason, Caitlyn, Gilbert, Wendy V
• Format: Journal Article
• Language: English
• Published: 01-04-2017
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.31.1_supplement.595.3

Abstract only RNA is extensively chemically modified in all organisms. These modifications were thought to be restricted to stable non‐coding RNAs, however recent work has revealed the presence of a variety of post‐transcriptional modifications in mRNA. Our lab recently developed Pseudo‐seq, a transcriptome‐wide approach to pseudouridine identification, which led to the discovery of widespread pseudouridylation of mRNAs in yeast, mouse, and human cells. We found that mRNA pseudouridylation events are regulated in response to cellular growth state; however, little is known about the mechanisms responsible for selective, regulated targeting of specific sequences for modification. We have sought to understand the mechanisms underlying how the conserved pseudouridine synthase Pus1 recognizes its mRNA targets. We have reconstituted Pus1‐dependent modification of synthetic mRNAs in vitro. By computational prediction, high‐throughput RNA structure probing, and mutational analysis we have revealed a conserved RNA structure that is necessary and sufficient for mRNA pseudouridylation by Pus1. These results provide insight into how mRNA targets are recognized by pseudouridine synthases, and our experimental approach is applicable to the study of all enzyme‐mRNA interactions.
Support or Funding Information
This work was supported by grants from The American Cancer Society – Robbie Sue Mudd Kidney Cancer Research Scholar Grant (RSG‐13‐396‐01‐RMC) and the National Institutes of Health (GM094303, GM081399) to W.V.G. T.M.C. was supported by the American Cancer Society New England Division (Ellison Foundation Postdoctoral Fellowship). This work was supported in part by the NIH Pre‐Doctoral Training Grant T32GM007287.