Targeted pseudouridylation identifies a novel Ψ-Ψ codon-anticodon pairing in nonsense suppression and translational recoding
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1273852 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR33969878.fastq.gz | Fastqsanger.gz | |||
| SRR33969879.fastq.gz | Fastqsanger.gz | |||
| SRR33969880.fastq.gz | Fastqsanger.gz | |||
| SRR33969881.fastq.gz | Fastqsanger.gz | |||
| SRR33969882.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 5 of 8 |
Similar Datasets
Project description:Pseudouridine (Ψ) is known for decades, but its flexibility in base-pairing remains unclear. This study engineers artificial box H/ACA guide RNAs to direct pseudouridylation at the uridine of a premature termination codon (PTC; UAA, UAG, or UGA) within an intron-less mRNA and U36 of the anticodon of a matching tRNA in yeast and human cells. Targeted pseudouridylation leads to the formation of a Ψ-Ψ codon-anticodon pair, which, together with the other two Watson-Crick base pairs in the codon-anticodon duplex, significantly improves codon-anticodon recognition, robustly promoting PTC read-through. The intron-less mRNA level remains unchanged with or without guide RNAs. Additionally, pseudouridylation does not impact tRNA stability or charging. Our results show that nonsense suppression is promoted by the high affinity of the Ψ-Ψ pair, which is verified by melting curve analysis. This work has identified an unusual Ψ-Ψ base pair, which contributes significantly to codon-anticodon recognition and translational recoding.
2025-06-20 | GSE299290 | 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: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: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 (psi) is one of the most abundant human mRNA modifications generated via psi synthases, including TRUB1 and PUS7. Nanopore direct RNA sequencing combined with our recent tool, Mod-p ID, enables psi mapping, transcriptome-wide, without chemical derivatization of the input RNA and/or conversion to cDNA. This method is sensitive for detecting changes in positional psi occupancies across cell types, which can inform our understanding of the impact on gene expression. We sequenced, mapped, and compared the positional psi occupancy across six immortalized human cell lines derived from diverse tissue types. We found that lung-derived cells have the highest proportion of psi, while liver-derived cells have the lowest. Further, we find that conserved psi positions correspond to higher levels of protein expression than expected, suggesting translation regulation. Interestingly, we identify cell type-specific sites of psi modification in ubiquitously expressed genes. Finally, we characterize sites with multiple psi modifications on the same transcript and found that these can be conserved or cell type specific, including examples of multiple psi modifications within the same k-mer. Our data support the hypothesis that psi modifications contribute to regulating translation and that cell type-specific trans-acting factors play a major role in driving pseudouridylation.
2024-08-20 | MSV000095655 | MassIVE
Project description:Pseudouridine (psi) is one of the most abundant human mRNA modifications generated via psi synthases, including TRUB1 and PUS7. Nanopore direct RNA sequencing combined with our recent tool, Mod-p ID, enables psi mapping, transcriptome-wide, without chemical derivatization of the input RNA and/or conversion to cDNA. This method is sensitive for detecting changes in positional psi occupancies across cell types, which can inform our understanding of the impact on gene expression. We sequenced, mapped, and compared the positional psi occupancy across six immortalized human cell lines derived from diverse tissue types. We found that lung-derived cells have the highest proportion of psi, while liver-derived cells have the lowest. Further, we find that conserved psi positions correspond to higher levels of protein expression than expected, suggesting translation regulation. Interestingly, we identify cell type-specific sites of psi modification in ubiquitously expressed genes. Finally, we characterize sites with multiple psi modifications on the same transcript and found that these can be conserved or cell type specific, including examples of multiple psi modifications within the same k-mer. Our data support the hypothesis that psi modifications contribute to regulating translation and that cell type-specific trans-acting factors play a major role in driving pseudouridylation.
2024-08-22 | MSV000095673 | MassIVE
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
Project description:edu-Ψ-seq Reveals snoRNA-guided mRNA Pseudouridylation that Regulates Protein Synthesis
| PRJNA788526 | ENA
Project description:Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ ‘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 increased protein biosynthesis and abnormal germ layer specification. Remarkably, dysregulation of PUS7 and tRFs in myeloid malignancies associates with altered translation rates, suggesting a role of Ψ in leukemogenesis. Our findings unveil a critical function of Ψ in directing translational control in stem cells with important implications for human disease.
2018-04-11 | PXD008676 | Pride