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Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing

ABSTRACT: The abundant RNA modification pseudouridine (Ψ) has been mapped transcriptome-wide by chemically modifying pseudouridines with carbodiimide and detecting the resulting reverse transcription stops in high-throughput sequencing. However, these methods have limited sensitivity and specificity, in part due to the use of reverse transcription stops. We sought to use mutations rather than just stops in sequencing data to identify pseudouridine sites. Here, we identify reverse transcription conditions that allow read-through of carbodiimide-modified pseudouridine (CMC-Ψ), and we show that pseudouridines in carbodiimide-treated human ribosomal RNA have context-dependent mutation and stop rates in high-throughput sequencing libraries prepared under these conditions. Furthermore, accounting for the context-dependence of mutation and stop rates can enhance the detection of pseudouridine sites. Similar approaches could contribute to the sequencing-based detection of many RNA modifications.

ORGANISM(S): Homo sapiens

PROVIDER: GSE110247 | GEO | 2018/05/07

REPOSITORIES: GEO

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Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution

Project description:Functional characterization of pseudouridine (Ψ) in mammalian messenger RNA (mRNA) has been hampered by the lack of a quantitative method that maps pseudouridine in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq) that utilizes a bisulfite-mediated reaction to stoichiometrically convert pseudouridine into deletion upon reverse transcription. BID-seq enabled detection of abundant pseudouridine sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10-20 ng input RNA. We uncovered consensus sequences for pseudouridine in mammalian mRNA and assigned different ‘writer’ proteins to individual pseudouridine deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detected presence of Ψ within stop codons of mammalian mRNA, and confirmed the role of Ψ in promoting stop codon read-through in vivo. This new method for sensitive and comprehensive detection of Ψ sets the stage for future investigations of the roles of Ψ in diverse biological processes.

2022-09-05 | GSE179798 | GEO

RNA Post-transcriptional Modifications in Two Large Subunit Intermediates Populated in E.coli Cells Expressing Helicase Inactive R331A DbpA

Project description:23S ribosomal RNA (rRNA) of Escherichia coli 50S large ribosome subunit contains 26 post-transcriptionally modified nucleosides. Here, we determine the extent of modifications in the 35S and 45S large subunit intermediates, accumulating in cells expressing the helicase inactive DbpA protein, R331A, and the native 50S large subunit. The modifications we characterized are nine pseudouridines, 3-methylpseudouridine, 2-methyladenine, and 5-hydroxycytosine. These modifications were detected using 1-cyclohexyl-(2-morpholinoethyl)carbodiimide metho-p-toluene sulfonate (CMCT) treatment followed by alkaline treatment. In addition, KMnO4 treatment of 23S rRNA was employed to detect 5-hydroxycytosine modification. CMCT and KMnO4 treatment produce chemical changes in modified nucleotides that cause reverse transcriptase misincorporations and deletions, which were detected employing next generation sequencing. Our results show that seven uridines to pseudouridine isomerizations and the 2-methyladenosine modification are present both in the 35S and 45S to similar extents as in the 50S. Hence, the enzymes that perform these modifications, namely RluF, RluB, RlmN, RluA, RluC, and RluA, have already acted in the intermediates. While two uridines to pseudouridines isomerizations, the 3-methylpseudouridine, and 5-hydroxycytosine modifications are significantly less present in the 35S and 45S as compared to the 50S. Therefore, the enzymes that perform these modifications, RluD, RlmH, and RlhA, are in the process of modifying the 35S, and 45S or these enzymes act during the later stages of ribosome assembly. Our study employs a novel high throughput and single nucleotide resolution technique for detection of 2-methyladenine and two novel high throughput and single nucleotide resolution technique for detection of 5-hydroxycytosine.

2022-09-15 | GSE196821 | GEO

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

2014-08-24 | GSE60445 | GEO

A radiolabeling-free, qPCR-based method for locus-specific pseudouridine detection

Project description:Pseudouridine (Ψ) is the most abundant post-transcriptional RNA modification. Various methods have been developed to achieve locus-specific Ψ detection; however, the existing methods often involve radiolabeling of RNA, require advanced experimental skills and can be time-consuming. Herein we report a radiolabeling-free，qPCR-based method to detect locus-specific Ψs in rRNA and mRNA. This method is based on Ψ chemical adduct (Ψ-CMC) induced mutation/deletion during reverse transcription (RT), leading to qPCR products of different melting temperatures. Utilizing high-throughput sequencing, we demonstrate that such misincorporation is a general feature of Ψ-CMC adduct during our improved RT conditions. We validated this method on known Ψ sites in rRNA and showed that the melting curves correlate with the modification level. Moreover, we successfully detected Ψs in mRNA and lncRNA of different abundance, and identified Ψ synthase that targets mRNA. Our facile method takes only 1.5 days to complete, and with slight adjustment it can be applied to detect other epitranscriptomic marks in the transcriptome.

2017-10-03 | GSE102476 | 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

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

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

Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing

Project description:Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing

| PRJNA433264 | ENA

A new reagent for in vivo structure probing of RNA G and U residues that improves RNA structure prediction alone and combined with DMS

Project description:A key to understanding the roles of RNA in regulating gene expression is knowing their structures in vivo. One way to obtain this information is through probing structures of RNA with chemicals. To probe RNA structure directly in cells, membrane-permeable reagents that modify the Watson-Crick (WC) face of unpaired nucleotides can be used. While dimethyl sulfate (DMS) has led to substantial insight into RNA structure, it has limited nucleotide specificity in vivo, with WC face reactivity only at Adenine (A) and Cytosine (C) at neutral pH. The reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was recently shown to modify the WC face of Guanine (G) and Uracil (U). While useful at lower concentrations in experiments that measure chemical modifications by reverse transcription stops, at higher concentrations necessary for detection by mutational profiling (MaP), EDC treatment leads to degradation of RNA. Indeed, herein we demonstrate EDC-stimulated degradation of RNA in Gram-negative and Gram-positive bacteria. In an attempt to overcome these limitations, we developed a new carbodiimide reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide (ETC), which we show specifically modifies unpaired Gs and Us in vivo without substantial degradation of RNA. We establish ETC as a probe for MaP and optimize the reverse transcription conditions and computational analysis in Escherichia coli. Importantly, we demonstrate the utility of ETC as a probe for improving RNA structure prediction both alone and with DMS.

2024-04-09 | GSE254895 | 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

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