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Updated: Nov 3, 2025

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity
Published on: October 8, 2018
Investigating Pseudouridylation Mechanisms by High-Throughput in Vitro RNA Pseudouridylation and Sequencing
Nicole M Martinez1, Wendy V Gilbert2
1Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
Abstract:
Pseudouridine profiling has revealed many previously unknown sites of the RNA modification pseudouridine (Ψ) in cellular RNAs. All organisms express multiple pseudouridine synthases (PUS) whose RNA targets and mechanisms of targeting remain to be elucidated. Here, we describe a high-throughput in vitro pseudouridylation assay to interrogate pseudouridine status upon incubation with recombinant pseudouridine synthases (PUS) at thousands of RNA sequences of interest in parallel. This approach allows validation of sites provisionally identified in cells, identification of the direct targets of individual PUS, and interrogation of the determinants of target recognition including primary sequence and RNA secondary structure.
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