{"database":"biostudies-literature","file_versions":[],"scores":null,"additional":{"submitter":["Martinez Campos C"],"funding":["Center for HIV RNA Studies","AIDS Reagent Program, Division of AIDS, NIAID, NIH","NIDA NIH HHS","NIAID NIH HHS","Duke University School of Medicine for the use of the Proteomics and Metabolomics Shared Resource","UPLC-MS/MS services","National Institutes of Health","Duke University Center for AIDS Research","NIH","Mexican National Science and Technology Council"],"pagination":["1400-1411"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/S-EPMC8522693"],"repository":["biostudies-literature"],"omics_type":["Unknown"],"volume":["27(11)"],"pubmed_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."],"journal":["RNA (New York, N.Y.)"],"pubmed_title":["Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique."],"pmcid":["PMC8522693"],"funding_grant_id":["R01 DA046111","R01-DA046111","U54 AI150470","P30 AI064518","711023/740541","P30-AI064518","U54-AI15047"],"pubmed_authors":["Courtney DG","Cullen BR","Holley CL","Tsai K","Bogerd HP","Martinez Campos C"],"additional_accession":[]},"is_claimable":false,"name":"Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique.","description":"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.","dates":{"release":"2021-01-01T00:00:00Z","publication":"2021 Nov","modification":"2026-06-12T09:57:22.172Z","creation":"2025-04-06T12:10:42.406Z"},"accession":"S-EPMC8522693","cross_references":{"pubmed":["34376564"],"doi":["10.1261/rna.078940.121"]}}