Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: 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.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE63655 | GEO | 2015/06/05

SECONDARY ACCESSION(S): PRJNA268578

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

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

Global PUS1 mRNA targets detection by STAMP

Project description:Recently, several studies revealed that pseudourine exists on mRNA. However, identification of ψ sites for specific PUS remains a certain ambiguity. PUS1, an important pseudouridine synthase, plays a critical role in cell growth and tumorigenesis of HCC. Here, we utilized an IP-free approach, Surveying Targets by APOBEC-Mediated Profiling (STAMP) and analyzed C-to-U editing sites to realize PUS1-bound mRNAs. And then, we identified the potential mRNA pseudouridylation targets of PUS1 by intersecting editing sites’ host genes with these that known to pseudouridylated.

2024-02-26 | GSE229980 | GEO

Quantitative profiling of pseudouridylation landscape in the human transcriptome

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

The molecular basis of tRNA selectivity by human pseudouridine synthase 3 (PUS3)

Project description:Pseudouridine (Ψ), the isomer of uridine, is ubiquitous in most RNA families, including tRNA, rRNA and mRNA. Pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs, but whether it can modify other RNA types, including mRNA, remains elusive. Here, we determine the single particle cryo-EM structure of apo and tRNA-bound human PUS3, showing how it forms a symmetric homodimer that recognizes the characteristic L-shape of tRNA across two distinct interaction interfaces. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and used Pseudo-seq to detect Ψ sites in the transcriptome. Although PUS1-dependent sites were detectable in tRNA and mRNA, we did not find any evidence for PUS3 modifying other RNA classes than tRNA. In summary, our work provides the molecular basis for PUS3-mediated tRNA modification in humans and aids in understanding how impairments in its activity lead to intellectual disabilities through defects in tRNA modifications.

2024-07-10 | GSE255287 | GEO

edu-Ψ-seq Reveals snoRNA-guided mRNA Pseudouridylation that Regulates Protein Synthesis [CLIP-seq]

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

edu-Ψ-seq Reveals snoRNA-guided mRNA Pseudouridylation that Regulates Protein Synthesis

2024-10-18 | GSE190820 | GEO

Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

Project description:Pseudouridine (Ψ) is one of the most abundant modifications in cellular RNA. However, its function remains elusive, mainly due to the lack of highly sensitive and accurate detection methods. To address this challenge, we introduced 2-bromoacrylamide-assisted cyclization sequencing (BACS) for quantitative profiling of Ψ at single-base resolution. Based on novel bromoacrylamide cyclization chemistry, BACS enables a Ψ-to-C transition. Compared to previous methods, BACS allowed the precise identification of Ψ positions, especially in densely modified Ψ regions and consecutive uridine sequences. BACS successfully detected all known Ψ sites in human rRNA and spliceosomal snRNAs and generated the first quantitative Ψ map of human snoRNA and tRNA. Furthermore, BACS simultaneously detected adenosine-to-inosine (A-to-I) editing sites and N1-methyladenosine (m1A). Depletion of three key pseudouridine synthases (PUS) enabled us to elucidate the targets and sequence motifs of TRUB1, PUS7, and PUS1 in HeLa cells. We further applied BACS to Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) and identified a highly abundant Ψ114 site in EBER2. Surprisingly, applying BACS to a panel of RNA viruses demonstrated the absence of Ψ in their viral transcripts or genomes, shedding light on differences in pseudouridylation between virus families. We anticipate BACS to serve as a powerful tool to uncover the biological importance of Ψ in future studies.

2024-07-31 | GSE241849 | GEO

Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique

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.

2021-09-01 | GSE172136 | GEO

Saccharomyces cerevisiae

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

| PRJNA258278 | ENA

A systematic dissection of determinants and consequences of snoRNA-guided pseudouridylation of human mRNA

Project description:RNA can be extensively modified post-transcriptionally with >170 covalent modifications, expanding its functional and structural repertoire. Pseudouridine (Ψ), the most abundant modified nucleoside in rRNA and tRNA, has recently been found within mRNA molecules. Due to technical challenges, it remained unclear whether pseudouridylation of mRNA can be snoRNA-guided, which has important implications for understanding the physiological target spectrum of snoRNAs and for their potential therapeutic exploitation in genetic diseases. Here, using a massively parallel reporter based strategy we simultaneously interrogate Ψ levels across hundreds of synthetic constructs with predesigned complementarity against endogenous snoRNAs. Our results demonstrate that snoRNA-mediated pseudouridylation can occur on mRNA targets. However, this is typically achieved at low efficiencies, and is constrained by the localization of mRNA, by the expression levels of snoRNAs and by the length of the snoRNA:mRNA complementarity stretches. We exploited these insights for the design of snoRNAs targeting pseudouridylation at premature termination codons, which was previously suggested to suppress translational termination. However, in contrast to previous studies, in this and follow-up experiments we observe no evidence for readthrough of pseudouridylated stop codons. Our study enhances our understanding of the scope, ‘design rules’ and constraints of snoRNA-mediated pseudouridylation, and challenges a key functional outcome associated with this modification.

2024-06-02 | GSE159749 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data