Mapping of pseudouridine residues on viral and cellular transcripts using a novel antibody-based technique

Cecilia Martinez Campos, Kevin Tsai, David G Courtney, Hal P Bogerd, Christopher L Holley, Bryan R Cullen

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Pseudouridine (ψ) is the most common non-canonical ribonucleoside present on mammalian non-coding RNAs (ncRNAs), including rRNAs, tRNAs and snRNAs, where it contributes ~7% of the total uridine level. However, ψ constitutes only ~0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear. Ψ residues have been shown to inhibit the detection of exogenous RNA transcripts by host innate immune factors, thus raising the possibility that viruses might have subverted the addition of ψ residues to mRNAs by host pseudouridine synthase (PUS) enzymes as a way to inhibit antiviral responses in infected cells. Here, we describe and validate a novel antibody-based ψ mapping technique called photo-crosslinking assisted ψ sequencing (PA-ψ-seq) and use it to map ψ residues on not only multiple cellular RNAs but also on the mRNAs and genomic RNA encoded by HIV-1. We describe 293T-derived cell lines in which human PUS enzymes previously reported to add ψ residues to human mRNAs, specifically PUS1, PUS7 and TRUB1/PUS4, were inactivated by gene editing. Surprisingly, while this allowed us to assign several sites of ψ addition on cellular mRNAs to each of these three PUS enzymes, ψ sites present on HIV-1 transcripts remained unaffected. Moreover, loss of PUS1, PUS7 or TRUB1 function did not significantly reduce the level of ψ residues detected on total human mRNA below the ~0.1% level seen in wild type cells, thus implying that the PUS enzyme(s) that adds the bulk of ψ residues to human mRNAs remains to be defined.
Original languageEnglish
Pages (from-to)1400–1411
Issue number11
Early online date10 Aug 2021
Publication statusPublished - 10 Nov 2021


  • HIV-1
  • pseudouridine
  • post-transcriptional gene regulation
  • Epitranscriptomics


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