Pseudouridine profiling reveals widespread regulated mRNA pseudouridylation in yeast and human cells
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA251554 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR1327190.fastq.gz | Fastqsanger.gz | |||
| SRR1327200.fastq.gz | Fastqsanger.gz | |||
| SRR1327210.fastq.gz | Fastqsanger.gz | |||
| SRR1327220.fastq.gz | Fastqsanger.gz | |||
| SRR1327230.fastq.gz | Fastqsanger.gz |
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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 M-NM-(-seq, a method for transcriptome-wide quantitative mapping of pseudouridine. We validate M-NM-(-seq with synthetic spike-ins and de novo identification of the vast majority of previously reported pseudouridylated positions. M-NM-(-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 M-bM-^@M-^S 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. Examination of m6A methylation in human Hek293 and A549 cell lines, in human embryonic stem cells (ESCs) undergoing differentiation to neural progenitor cells (NPCs), in OKMS inducible fibroblasts reprogrammed into iPSC, and upon knockdown of factors using siRNAs or shRNAs.
2014-09-11 | E-GEOD-60047 | biostudies-arrayexpress
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
Project description:Transcriptome-wide mapping reveals widespread dynamic regulated pseudouridylation of mRNA
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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. CMC treatment of RNA followed by next generation sequencing was used to measure the extent of pseudouridylation at mapped mouse rRNA Ψ sites.
2016-08-07 | E-GEOD-78063 | biostudies-arrayexpress
Project description:Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function--it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1-4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.
| S-EPMC4224642 | biostudies-literature
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.
2023-06-26 | GSE211201 | GEO
Project description:Pseudouridylation (pseudouridine) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of pseudouridine remains poorly understood. Here, we show that a pseudouridine-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the pseudouridine ‘writer’ PUS7 modifies and activates a network of tRNA-derived fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translational regulation leading to high protein biosynthesis and abnormal germ layer specification. Dysregulation of PUS7 and tRFs in myeloid malignancies associates with altered translation rates, suggesting a role of pseudouridine in tumorigenesis. Our findings unveil a critical function of pseudouridine in directing translational control in stem cells with promisingly broad implications for human disease.
2018-04-09 | GSE101485 | GEO
Project description: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. RNA was treated with CMC to modify Ψ 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
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.
2023-06-26 | GSE211199 | GEO
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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