Investigating Pseudouridylation Mechanisms by High-Throughput in Vitro RNA Pseudouridylation and Sequencing

Nicole M Martinez; Wendy V Gilbert

doi:10.1007/978-1-0716-1374-0_22

Investigating Pseudouridylation Mechanisms by High-Throughput in Vitro RNA Pseudouridylation and Sequencing

Methods Mol Biol. 2021:2298:379-397. doi: 10.1007/978-1-0716-1374-0_22.

Authors

Nicole M Martinez¹, Wendy V Gilbert²

Affiliations

¹ Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
² Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA. wendy.gilbert@yale.edu.

PMID: 34085256
DOI: 10.1007/978-1-0716-1374-0_22

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.

Keywords: PUS; Pseudo-seq; Pseudouridine; Pseudouridine synthase; Pseudouridylation; RNA modification; mRNA modification.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Base Sequence / genetics
High-Throughput Nucleotide Sequencing / methods*
Intramolecular Transferases / genetics
Pseudouridine / genetics*
RNA / genetics*
RNA Processing, Post-Transcriptional / genetics

Substances

Pseudouridine
RNA
Intramolecular Transferases
pseudouridine synthases

Abstract

Publication types

MeSH terms

Substances

Grants and funding