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

Part of the Methods in Molecular Biology book series (MIMB,volume 2298)

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.

Key words

  • Pseudouridine
  • RNA modification
  • mRNA modification
  • PUS
  • Pseudouridine synthase
  • Pseudouridylation
  • Pseudo-seq

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  • DOI: 10.1007/978-1-0716-1374-0_22
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Acknowledgments

This work was supported by NIH (R01GM101316) to W.V.G. and a Jane Coffin Childs Postdoctoral Fellowship 161624T to N.M.M.

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Correspondence to Wendy V. Gilbert .

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Martinez, N.M., Gilbert, W.V. (2021). Investigating Pseudouridylation Mechanisms by High-Throughput in Vitro RNA Pseudouridylation and Sequencing. In: McMahon, M. (eds) RNA Modifications. Methods in Molecular Biology, vol 2298. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1374-0_22

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  • DOI: https://doi.org/10.1007/978-1-0716-1374-0_22

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  • Publisher Name: Humana, New York, NY

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