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

Publication ,  Journal Article
Martinez Campos, C; Tsai, K; Courtney, DG; Bogerd, HP; Holley, CL; Cullen, BR
Published in: RNA
November 2021

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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Published In

RNA

DOI

EISSN

1469-9001

Publication Date

November 2021

Volume

27

Issue

11

Start / End Page

1400 / 1411

Location

United States

Related Subject Headings

  • RNA, Viral
  • RNA, Messenger
  • RNA Processing, Post-Transcriptional
  • Pseudouridine
  • Intramolecular Transferases
  • Hydro-Lyases
  • Humans
  • HIV-1
  • HIV Infections
  • HEK293 Cells
 

Citation

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Martinez Campos, C., Tsai, K., Courtney, D. G., Bogerd, H. P., Holley, C. L., & Cullen, B. R. (2021). Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique. RNA, 27(11), 1400–1411. https://doi.org/10.1261/rna.078940.121
Martinez Campos, Cecilia, Kevin Tsai, David G. Courtney, Hal P. Bogerd, Christopher L. Holley, and Bryan R. Cullen. “Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique.RNA 27, no. 11 (November 2021): 1400–1411. https://doi.org/10.1261/rna.078940.121.
Martinez Campos C, Tsai K, Courtney DG, Bogerd HP, Holley CL, Cullen BR. Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique. RNA. 2021 Nov;27(11):1400–11.
Martinez Campos, Cecilia, et al. “Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique.RNA, vol. 27, no. 11, Nov. 2021, pp. 1400–11. Pubmed, doi:10.1261/rna.078940.121.
Martinez Campos C, Tsai K, Courtney DG, Bogerd HP, Holley CL, Cullen BR. Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique. RNA. 2021 Nov;27(11):1400–1411.

Published In

RNA

DOI

EISSN

1469-9001

Publication Date

November 2021

Volume

27

Issue

11

Start / End Page

1400 / 1411

Location

United States

Related Subject Headings

  • RNA, Viral
  • RNA, Messenger
  • RNA Processing, Post-Transcriptional
  • Pseudouridine
  • Intramolecular Transferases
  • Hydro-Lyases
  • Humans
  • HIV-1
  • HIV Infections
  • HEK293 Cells