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Co-transcriptional pseudouridylation of mRNAs by the H/ACA complex controls translational efficiency [RNA-seq]

ABSTRACT: Post-transcriptional modifications of mRNA have emerged as novel regulators of gene expression. Pseudouridylation is the most abundant and widespread type of RNA modification in living organisms; however, the biological role of pseudouridine in mRNAs remains poorly understood. Here, we show that the pseudouridine synthase dyskerin associates with RNA polymerase II and is enriched at RNA polymerase II-transcribed genes genome-wide. As part of the H/ACA complex, dyskerin binds to thousands of mRNAs and is responsible for their pseudouridylation, an action that occurs in chromatin and does not appear to require a fully complementary guide RNA. In cells lacking dyskerin, pseudouridylation of mRNAs is reduced, while at the same time, de novo protein production is enhanced, indicating that pseudouridylation of mRNAs by dyskerin interferes with translation. Accordingly, pseudouridylation of an mRNA by dyskerin in vitro results in reduced translation of that mRNA. Moreover, in patients with dyskeratosis congenita caused by inherited mutations in the DKC1 gene, binding between mutated dyskerin and mRNAs is altered and pseudouridylation of mRNAs severely reduced. Our findings reveal a new critical function of the H/ACA complex in directing translation control with important implications for development and disease.

ORGANISM(S): Homo sapiens

PROVIDER: GSE211200 | GEO | 2023/06/26

REPOSITORIES: GEO

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Co-transcriptional pseudouridylation of mRNAs by the H/ACA complex controls translational efficiency [iCLIP]

Project description:Post-transcriptional modifications of mRNA have emerged as novel regulators of gene expression. Pseudouridylation is the most abundant and widespread type of RNA modification in living organisms; however, the biological role of pseudouridine in mRNAs remains poorly understood. Here, we show that the pseudouridine synthase dyskerin associates with RNA polymerase II and is enriched at RNA polymerase II-transcribed genes genome-wide. As part of the H/ACA complex, dyskerin binds to thousands of mRNAs and is responsible for their pseudouridylation, an action that occurs in chromatin and does not appear to require a fully complementary guide RNA. In cells lacking dyskerin, pseudouridylation of mRNAs is reduced, while at the same time, de novo protein production is enhanced, indicating that pseudouridylation of mRNAs by dyskerin interferes with translation. Accordingly, pseudouridylation of an mRNA by dyskerin in vitro results in reduced translation of that mRNA. Moreover, in patients with dyskeratosis congenita caused by inherited mutations in the DKC1 gene, binding between mutated dyskerin and mRNAs is altered and pseudouridylation of mRNAs severely reduced. Our findings reveal a new critical function of the H/ACA complex in directing translation control with important implications for development and disease.

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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.

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Project description:Co-transcriptional pseudouridylation of mRNAs by the H/ACA complex controls translational efficiency.

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