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Exonuclease-assisted identification of pseudouridine sites in single-stranded RNA

ABSTRACT: Exonuclease-assisted identification of pseudouridine sites in single-stranded RNA

PROVIDER: PRJNA954003 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR24120246_1.fastq.gz	Fastqsanger.gz
	SRR24120246_2.fastq.gz	Fastqsanger.gz
	SRR24120247_1.fastq.gz	Fastqsanger.gz
	SRR24120247_2.fastq.gz	Fastqsanger.gz
	SRR24120248_1.fastq.gz	Fastqsanger.gz

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Exonuclease-assisted identification of pseudouridine sites in single-stranded RNA

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Project description:Pseudouridine is an isomer of uridine and is the most common RNA modification in both procaryotes and eucaryotes. It is found in ribosomal, transfer, and other structural RNA as well as in some mRNA and non-coding RNA. We have found abundant pseudouridine in small RNA and their precursors in Arabidopsis.

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Project description:Pseudouridine (ψ) is the most common non-canonical ribonucleoside present on mammalian non-coding RNAs (ncRNAs), where is contributes ~10% of the total uridine level. However, ψ constitutes only ~0.3% of the uridines present on mRNAs and its effect on mRNA function remains unclear. Ψ residues have however 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 then use it to map ψ residues on not only multiple cellular RNAs but also the mRNAs and genomic RNA encoded by HIV-1. We also describe several 293T-derived cell lines in which human PUS enzymes previously reported to add ψ residues to mRNAs, specifically PUS1, PUS7 and TRUB1/PUS4, were individually 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, the ψ sites present on HIV-1 transcripts remained unaffected. However, loss of PUS1 function did significantly reduce HIV-1 gene expression by a presumably indirect mechanism.

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