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Pseudouridine profiling reveals widespread regulated mRNA pseudouridylation in yeast and human cells

ABSTRACT: We report the development of Pseudo-Seq, a sequencing based technique for identification of pseuoduridine (Ψ) modifications within RNA. We performed Pseudo-Seq on S. cerevisiae and HeLa cells and find the first evidence for pseusouridylation of endogenous mRNAs.

ORGANISM(S): Homo sapiens Saccharomyces cerevisiae

PROVIDER: GSE58200 | GEO | 2014/09/05

SECONDARY ACCESSION(S): PRJNA251554

REPOSITORIES: GEO

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Pseudouridine profiling reveals widespread regulated mRNA pseudouridylation in yeast and human cells

Project description:We report the development of Pseudo-Seq, a sequencing based technique for identification of pseuoduridine (Î¨) modifications within RNA. We performed Pseudo-Seq on S. cerevisiae and HeLa cells and find the first evidence for pseusouridylation of endogenous mRNAs. RNA was treated with CMC to modify Î¨ residues, which cause RT stops. The sites of CMC-dependent RT stops were used to identify sites of pseudouridyation.

2014-09-05 | E-GEOD-58200 | biostudies-arrayexpress

Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells.

Project description: Not available

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Transcriptome-wide mapping reveals widespread dynamic regulated pseudouridylation of mRNA

Project description:Pseudouridine is the most abundant modification occurring on RNA, yet with the exception of a few well-studied RNA molecules little is known about the modified positions and their function(s). Here, we develop Ψ-seq, a method for transcriptome-wide quantitative mapping of pseudouridine. We validate Ψ-seq with synthetic spike-ins and de novo identification of the vast majority of previously reported pseudouridylated positions. Ψ-seq permits discovery of hundreds of novel pseudouridine modifications in human and yeast mRNAs and snoRNAs. Knockdown and knockout of pseudouridine synthases uncovers the cognate PUSs mediating pseudouridine catalysis at these individual novel sites and their target sequence features. In both human and yeast pseudouridine formation on mRNA depends on both site-specific PUSs – often guided by a specific sequence motif - and snoRNA-guided PUSs. Importantly, upon heat shock in yeast, Pus7-mediated pseudouridylation is induced at >200 sites in diverse mRNAs. Pus7 deletion in yeast leads to decreased recovery from heat shock and decreased RNA levels at otherwise pseudouridylated messages, suggesting a role for pseudouridine in enhancing transcript stability. Pseudouridine stoichiometries in rRNA are highly conserved from yeast to mammals, but are reduced in cells derived from dyskeratosis congenita patients, where the pseudouridine synthase DKC1 is mutated, compared to age matched controls. Our results establish pseudouridine as a ubiquitous and dynamic modification in mRNA, and provide a sensitive, quantitative and transcriptome-wide methodology to address its underlying mechanisms and function.

2014-09-11 | GSE60047 | GEO

Pseudouridylation in Naf1+/- mice

Project description:Pseudo-seq was used to measure differences in the extent of pseudouridine (Ψ) modification in rRNA from mice haploinsufficient for the H/ACA biogenesis factor Naf1 compared to wild- type. We measured telomerase RNA levels as well as a sample of box H/ACA RNAs and found they were all decreased in Naf1+/- mice. Nevertheless, Naf1+/- mice had minimally decreased levels of pseudouridylation at the rRNA sites queried. This was not associated with any phenotypic abnormalities in vivo in these mice which we examined for hematopoeitic, liver, testes and brain defects.

2016-08-07 | GSE78063 | GEO

Pseudouridine profiling reveals widespread regulated mRNA pseudouridylation in yeast and human cells

Project description:Pseudouridine profiling reveals widespread regulated mRNA pseudouridylation in yeast and human cells

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Yeast D10 Small ~ WT San I

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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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