Pseudouridylation in Naf1+/- mice
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE78063 | GEO | 2016/08/07
SECONDARY ACCESSION(S): PRJNA312494
REPOSITORIES: GEO
Similar Datasets
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:Pseudouridine (Ψ) is the most abundant RNA modification, yet little is known about its content, dynamics and function in mRNA and ncRNA. Here, we perform quantitative MS analysis and develop CAP-seq for transcriptome-wide Ψ profiling. The unexpected high Ψ content (Ψ/U ratio: ~ 0.2% to 0.6%) indicates that pseudouridylation in mammalian mRNA is much more prevalent and comprehensive than previously believed. In concordance, CAP-seq identified 2,084 Ψ sites within 1,929 human transcripts. We prove four previously unknown Ψ sites in rRNA and EEF1A1 mRNA. Genetic and biochemical analysis uncover PUS1 as a major human mRNA Ψ synthase. In response to stimuli, Ψ level and sites are dynamically modulated in stimulus-specific manners. Comparisons between human and mouse pseudouridylation reveal conserved and unique sites across tissue and species. We observe stop codon pseudouridylation and readthrough events simultaneously for HSPB1 mRNA, indicating a role in nonsense suppression. Together, these approaches allow in-depth analysis of transcriptome-wide pseudouridylation events and our comprehensive study provides a resource for functional studies of Ψ-mediated epigenetic regulation. Here we report a transcriptome-wide profiling method that utilizes a chemically synthesized N3-CMC, which pre-enriches the Ψ-containing RNAs and blocks the reverse transcription.Mapping the Ψ sites in human transcriptome was performed using HEK293T and PUS1 dependent Ψ sites were identified by comparing PUS1 knock out cells with wildtype cells. Stress inducible or suppressed Ψ sites were identified by comparing stress treated cells with untreated cells. And mouse brain and liver were used to map Ψ sites in mouse transcriptome.
2015-06-05 | E-GEOD-63655 | biostudies-arrayexpress
Project description:Pseudouridine (Ψ) is the most abundant RNA modification, yet little is known about its content, dynamics and function in mRNA and ncRNA. Here, we perform quantitative MS analysis and develop CAP-seq for transcriptome-wide Ψ profiling. The unexpected high Ψ content (Ψ/U ratio: ~ 0.2% to 0.6%) indicates that pseudouridylation in mammalian mRNA is much more prevalent and comprehensive than previously believed. In concordance, CAP-seq identified 2,084 Ψ sites within 1,929 human transcripts. We prove four previously unknown Ψ sites in rRNA and EEF1A1 mRNA. Genetic and biochemical analysis uncover PUS1 as a major human mRNA Ψ synthase. In response to stimuli, Ψ level and sites are dynamically modulated in stimulus-specific manners. Comparisons between human and mouse pseudouridylation reveal conserved and unique sites across tissue and species. We observe stop codon pseudouridylation and readthrough events simultaneously for HSPB1 mRNA, indicating a role in nonsense suppression. Together, these approaches allow in-depth analysis of transcriptome-wide pseudouridylation events and our comprehensive study provides a resource for functional studies of Ψ-mediated epigenetic regulation.
2015-06-05 | GSE63655 | GEO
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:Purpose:Pseudouridine (Ψ) is the most abundant RNA epigenetic modification and plays vital roles in various biological processes. However, its biogenesis and functions in mRNAs remain elusive. Method:Here, we developed an approach for enriching Ψ sites with deep sequencing (edu-Ψ-seq), which utilizes both exoribonuclease and restriction endonuclease activity to eliminate other nucleosides or reads without Ψ modifications. Results:Using edu-Ψ-seq approach in the dyskerin pseudouridine synthase 1 (DKC1) knockdown cells, we identified that more than 100 pseudouridines could be catalyzed by DKC1 in human mRNAs and non-coding RNAs (ncRNAs). Surprisingly, we discovered, for the first time, 10 pseudouridines in mRNAs might be guided by 11 small nucleolar ncRNAs (snoRNAs). Interestingly, we found that SNORA10 could guide DKC1 protein to induce the pseudouridylation of RPL15 mRNA. Importantly, loss of SNORA10 impaired the protein synthesis of RPL15 and inhibited cell proliferation. Conclusion:Overall, our study not only developed a powerful method for detecting Ψ sites but also uncovered another layer of gene expression regulation that involves crosstalk among snoRNAs, mRNA pseudouridylation and protein synthesis.
2024-10-18 | GSE190820 | GEO
Project description:Pseudouridine (Ψ) is an abundant post-transcriptional RNA modification in ncRNA and mRNA. However, transcriptome-wide measurement of individual Ψ sites remains unaddressed. Here, we develop “PRAISE”, via selective chemical labeling of Ψ by bisulfite to induce nucleotide deletion signature during reverse transcription, to realize quantitative assessment of the Ψ landscape in the human transcriptome. Unlike traditional RNA/DNA bisulfite treatment, our approach is based on quaternary base mapping and identifies 2,209 confident Ψ sites in HEK293T cells. By perturbing pseudouridine synthases, we obtained differential mRNA targets of PUS1, PUS7, TRUB1 and DKC1. In addition, we identified known and novel Ψ sites in mitochondrial mRNA, which are catalyzed by a mitochondria-localized isoform of PUS1. Collectively, we provide a reliable, sensitive and convenient method to quantify transcriptome-wide Ψ; we envision this approach would facilitate emerging efforts to elucidate the function and mechanism of mRNA pseudouridylation.
2023-02-06 | GSE212210 | GEO
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:Purpose:Pseudouridine (Ψ) is the most abundant RNA epigenetic modification and plays vital roles in various biological processes. However, its biogenesis and functions in mRNAs remain elusive. Method:Here, we developed an approach for enriching Ψ sites with deep sequencing (edu-Ψ-seq), which utilizes both exoribonuclease and restriction endonuclease activity to eliminate other nucleosides or reads without Ψ modifications. Results:Using edu-Ψ-seq approach in the dyskerin pseudouridine synthase 1 (DKC1) knockdown cells, we identified that more than 100 pseudouridines could be catalyzed by DKC1 in human mRNAs and non-coding RNAs (ncRNAs). Surprisingly, we discovered, for the first time, 10 pseudouridines in mRNAs might be guided by 11 small nucleolar ncRNAs (snoRNAs). Interestingly, we found that SNORA10 could guide DKC1 protein to induce the pseudouridylation of RPL15 mRNA. Importantly, loss of SNORA10 impaired the protein synthesis of RPL15 and inhibited cell proliferation. Conclusion:Overall, our study not only developed a powerful method for detecting Ψ sites but also uncovered another layer of gene expression regulation that involves crosstalk among snoRNAs, mRNA pseudouridylation and protein synthesis.
2024-10-18 | GSE192381 | 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.
2023-06-26 | GSE211201 | 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.
2023-06-26 | GSE211200 | GEO