Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique
Autor: | Bryan R. Cullen, Hal P. Bogerd, Cecilia Martínez-Campos, David G. Courtney, Christopher L. Holley, Kevin Tsai |
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Rok vydání: | 2021 |
Předmět: |
HIV Infections
Biology Pseudouridine 03 medical and health sciences chemistry.chemical_compound Epitranscriptomics Humans RNA Messenger RNA Processing Post-Transcriptional Intramolecular Transferases Molecular Biology Hydro-Lyases 030304 developmental biology Gene Editing chemistry.chemical_classification 0303 health sciences Messenger RNA 030302 biochemistry & molecular biology Wild type Antibodies Monoclonal RNA Ribonucleoside Uridine Cell biology HEK293 Cells Enzyme Biochemistry chemistry Cell culture HIV-1 RNA Viral Function (biology) |
Zdroj: | RNA. 27:1400-1411 |
ISSN: | 1469-9001 1355-8382 |
Popis: | Pseudouridine ({psi}) 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, {psi} constitutes only ~0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear. {Psi} 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 {psi} 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 {psi} mapping technique called photo-crosslinking assisted {psi} sequencing (PA-{psi}-seq) and use it to map {psi} residues on not only multiple cellular RNAs but also on the mRNAs and genomic RNA encoded by HIV-1. We describe several 293T-derived cell lines in which human PUS enzymes previously reported to add {psi} 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 {psi} addition on cellular mRNAs to each of these three PUS enzymes, the {psi} sites present on HIV-1 transcripts remained unaffected. Moreover, loss of PUS1, PUS7 or TRUB1 function did not significantly reduce the level of {psi} 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 {psi} residues to human mRNAs remains to be defined. |
Databáze: | OpenAIRE |
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