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

• Published in: RNA (Cambridge) Vol. 27; no. 11; pp. 1400 - 1411
• Main Authors: Martinez Campos, Cecilia, Tsai, Kevin, Courtney, David G, Bogerd, Hal P, Holley, Christopher L, Cullen, Bryan R
• Format: Journal Article
• Language: English
• Published: United States Cold Spring Harbor Laboratory Press 01-11-2021
• Subjects: Antibodies, Monoclonal - immunology; Cell lines; Enzymes; Gene Editing; Gene mapping; HEK293 Cells; HIV; HIV Infections - genetics; HIV Infections - metabolism; HIV Infections - virology; HIV-1 - physiology; Human immunodeficiency virus; Humans; Hydro-Lyases - antagonists & inhibitors; Hydro-Lyases - genetics; Hydro-Lyases - immunology; Hydro-Lyases - metabolism; Intramolecular Transferases - antagonists & inhibitors; Intramolecular Transferases - genetics; Intramolecular Transferases - immunology; Intramolecular Transferases - metabolism; Method; Pseudouridine - immunology; Pseudouridine - metabolism; Pseudouridine synthase; RNA Processing, Post-Transcriptional; RNA, Messenger - genetics; RNA, Messenger - metabolism; RNA, Viral - genetics; RNA, Viral - metabolism; Uridine; HIV-1; post-transcriptional gene regulation; pseudouridine; epitranscriptomics
• ISSN: 1355-8382; 1469-9001
• DOI: 10.1261/rna.078940.121

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.