Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique
- Cecilia Martinez Campos1,5,
- Kevin Tsai1,3,5,
- David G. Courtney1,4,
- Hal P. Bogerd1,
- Christopher L. Holley2 and
- Bryan R. Cullen1
- 1Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710, USA;
- 2Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA
- Corresponding author: bryan.cullen{at}duke.edu
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↵5 These authors contributed equally to this work.
Abstract
Pseudouridine (Ψ) is the most common noncanonical ribonucleoside present on mammalian noncoding 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.
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Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna.078940.121.
- Received August 4, 2021.
- Accepted August 4, 2021.
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