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Pseudouridylation in Naf1+/- mice

ABSTRACT: Pseudo-seq was used to measure differences in the extent of pseudouridine (Î¨) 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 Î¨ sites.

ORGANISM(S): Mus musculus

SUBMITTER: Thomas Carlile

PROVIDER: E-GEOD-78063 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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

Chemical Pull-Down Reveals Comprehensive and Dynamic Pseudouridylation in Mammalian Transcriptome

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

Regulated snoRNA expression during differentiation corresponds with a change in ribosomal RNA pseudouridylation and altered pre-ribosomal RNA processing [Nano77]

Project description:H/ACA small nucleolar RNAs (snoRNAs) guide pseudouridylation as part of a small nucleolar ribonucleoprotein complex (snoRNP). Disruption of H/ACA snoRNA expression in stem cells impairs pluripotency, yet it remains unclear how H/ACA snoRNAs contribute to differentiation. To determine if H/ACA snoRNA expression is dynamic during differentiation, we comprehensively profiled H/ACA snoRNA expression in multiple murine cell types and during differentiation in three cellular models, including mouse embryonic stem cells and mouse myoblasts. We determined that H/ACA snoRNA expression is cell-type specific, and we identified a subset of snoRNAs that are specifically regulated during differentiation. Additionally, we demonstrated that a decrease in Snora27 expression upon differentiation corresponds to a decrease in pseudouridylation of its target site within the E-site transfer RNA (tRNA) binding region of the 28S ribosomal RNA (rRNA) in the large ribosomal subunit. Many of the snoRNAs regulated during differentiation have target nucleotides in the 28S rRNA, and we found that pre-rRNA processing of large subunit precursors is altered during differentiation. Together, these data suggest a model in which H/ACA snoRNAs are specifically regulated during differentiation to potentially alter pseudouridylation and fine tune ribosome function.

2020-07-09 | GSE140622 | GEO

Regulated snoRNA expression during differentiation corresponds with a change in ribosomal RNA pseudouridylation and altered pre-ribosomal RNA processing [Nano104]

2020-07-09 | GSE140618 | GEO

Regulated snoRNA expression during differentiation corresponds with a change in ribosomal RNA pseudouridylation and altered pre-ribosomal RNA processing [RNA-seq and ncRNA-seq]

2020-07-09 | GSE139812 | GEO

PSI-seq to Identify Sites of Pseudouridylation in S. cerevisiae

Project description:Using a technique based on the ability of CMC to specifically label pseudouridines and to stop reverse transcriptase, we provide a transcriptome-wide map of pseudouridylation in S. cerevisiae under log phase and heat shock conditions 4 conditions: log phase (2x), heat shock, and stationary phase. For each condition, there are 4 libraries: natural RNA + or - CMC treatment and in vitro transcribed RNA + or - CMC treatment

2014-08-24 | E-GEOD-60445 | biostudies-arrayexpress

PSI-seq to Identify Sites of Pseudouridylation in S. cerevisiae

2014-08-24 | GSE60445 | GEO

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 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

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.

2023-06-26 | GSE211201 | GEO

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

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