The pseudouridine epitranscroptome of PA-ψ-seq HepAD38 tet- cell line
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE341816 | GEO | 2026/09/17
REPOSITORIES: GEO
Dataset's files
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| GSE341816_HBV_summits.bed.gz | Bed |
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Project description:Leishmania are unicellular parasites that cycle between the insect and mammalian host and is the causative agent of Leishmaniasis. Previous studies from our lab suggested that individual rRNA modification such as pseudouridine (Psi) and 2-O-methylation are developmentally regulated during the complex life cycle of these parasites. Ablation of a single snoRNA guiding Psi in the inter-subunit bridge, helix69 of the ribosome affects its affinity to translate specific mRNA. Mutational analysis in the pseudouridylation pocket of this snoRNA supports the notion that the guided modification and not the chaperone activity, is essential for the observed phenotype. Structural studies using CryoEM suggested that a single Psi modification can affect the rRNA structure. We propose that alteration in rRNA modifications could generate ribosomes preferentially translating state-beneficial proteins.
2024-05-09 | PXD034116 | Pride
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.
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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.
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Project description:Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that various viruses can manipulate the infection process by secretion of specific viral and cellular components into exosomes, small nanometer-sized (30-150 nm) vesicles secreted from various cells. However, the impact of HBV replication on hepatocytes produced exosomes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from conditioned medium of HepAD38 cell cultures and the purity of exosomes were confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC−MS/MS quantitation technologies were applied to analyze protein content of HBV-exosomes and HBV-free-exosomes. A total of 1412 exosomal proteins were identified, in which the abundance of 35 proteins were significantly altered. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HBV-exosomes. Bioinformatic analysis of differential exosomal proteins revealed the significant enrichment of components involved in proteasomal catabolic process. Proteasome activity assays further suggested that HBV-exosomes had enhanced proteolytic activity compared to HBV-free-exosomes. Furthermore, Human peripheral monocytes incubated with HBV-exosomes induced a significant lower level of IL-6 secretion compared to HBV-free-exosomes. Irreversible inhibition of proteasomal activity within exosome restored the IL-6 induction in monocytes. These results suggest that transmission of proteasome subunit proteins by HBV-exosomes might modulate the innate sensing of pro-inflammatory molecules in the recipient monocytes. These results revealed the composition and potential function of exosomes produced during HBV replication, thus provide a new perspective on the role of exosomes in HBV-host interaction.
2017-03-01 | PXD004724 | Pride
Project description:Pseudouridylation is a prevalent RNA modification shown to occur in tRNAs, rRNAs, snoRNAs and most recently mRNAs and ncRNAs. Emerging evidence suggests that this dynamic RNA modification is implicated in altering gene expression by regulating RNA stability, modulating translation elongation and modifying amino acid substitution rates. However, the role of pseudouridylation in infection is poorly understood. Here we demonstrate that Kaposi’s sarcoma-associated herpesvirus (KSHV) manipulates the pseudouriylation pathway to enhance replication. We show the pseudouridine synthases (PUS), PUS1 and PUS7 are essential for efficient KSHV lytic replication, supported by the redistribution of PUS7 to viral replication and transcription complexes. Here we present a comprehensive analysis of KSHV RNA pseudouridylation revealing hundreds of modified RNAs at single-nucleotide resolution. Notably, we further demonstrate that pseudouridylation of the KSHV-encoded polyadenylated nuclear RNA (PAN) plays a significant role in the stability of PAN RNA and in the association of the KSHV ORF57 protein. Our findings reveal a novel and essential role of pseudouridine modification in the KSHV replication cycle.
2025-11-05 | GSE217688 | 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 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
Project description:Hepatitis B virus (HBV) infection is a major health problem worldwide. Recent evidence suggests that various viruses can manipulate the infection process by secretion of specific viral and cellular components into exosomes, small nanometer-sized (30-150 nm) vesicles secreted from various cells. However, the impact of HBV replication on hepatocytes produced exosomes has not been fully delineated. In this work, an HBV-inducible cell line HepAD38 was used to directly compare changes in the protein content of exosomes secreted from HepAD38 cells with or without HBV replication. Exosomes were isolated from conditioned medium of HepAD38 cell cultures and the purity of exosomes were confirmed by transmission electron microscopy (TEM) and Western immunoblotting assays. Ion-intensity based label-free LC?MS/MS quantitation technologies were applied to analyze protein content of HBV-exosomes and HBV-free-exosomes. A total of 1412 exosomal proteins were identified, in which the abundance of 35 proteins were significantly altered. Strikingly, 5 subunit proteins from the 26S proteasome complex, including PSMC1, PSMC2, PSMD1, PSMD7 and PSMD14 were consistently enhanced in HBV-exosomes. Bioinformatic analysis of differential exosomal proteins revealed the significant enrichment of components involved in proteasomal catabolic process. Proteasome activity assays further suggested that HBV-exosomes had enhanced proteolytic activity compared to HBV-free-exosomes. Furthermore, Human peripheral monocytes incubated with HBV-exosomes induced a significant lower level of IL-6 secretion compared to HBV-free-exosomes. Irreversible inhibition of proteasomal activity within exosome restored the IL-6 induction in monocytes. These results suggest that transmission of proteasome subunit proteins by HBV-exosomes might modulate the innate sensing of pro-inflammatory molecules in the recipient monocytes. These results revealed the composition and potential function of exosomes produced during HBV replication, thus provide a new perspective on the role of exosomes in HBV-host interaction.
2017-03-29 | MSV000080718 | MassIVE
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