Nm-Mut-seq: a base-resolution quantitative method for mapping transcriptome-wide 2'-O-methylation.
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ABSTRACT:
SUBMITTER: Chen L
PROVIDER: S-EPMC10474006 | biostudies-literature | 2023 Sep
REPOSITORIES: biostudies-literature
Publications
Nm-Mut-seq: a base-resolution quantitative method for mapping transcriptome-wide 2'-O-methylation.
Chen Li L Zhang Li-Sheng LS Ye Chang C Zhou Huiqing H Liu Bei B Gao Boyang B Deng Zixin Z Zhao Changming C He Chuan C Dickinson Bryan C BC
Cell research 20230615 9
Similar Datasets
Project description:2’-O-methylation (Nm) is a prevalent post-transcriptional RNA modification present in many cellular RNAs and plays a critical role in modulating both the physical properties and regulation of eukaryotic RNAs. Studies of Nm modifications in RNA have long been hampered by a lack of effective mapping methods. Previously reported approaches can work well for detecting Nm modifications on abundant RNAs, but face challenges when applied to low-abundant RNAs, such as mRNA, lack stoichiometric information, and are challenged by issues of RNA sample degradation due to chemical treatment. Here, we present Nm-Mut-seq, a mutation signature-based Nm mapping method, which uses a custom reverse transcriptase (RT) that installs mutations at Am, Cm, and Gm-modified sites (Um is undetectable by this method). Our work provides a much-needed approach to detect Nm at base resolution in low abundant RNAs and to estimate the stoichiometry of each modified site transcriptome-wide.
2023-06-01 | GSE174518 | GEO
Project description:Nm-Mut-seq: The base-resolution quantitative method for mapping transcriptome-wide 2’-O-methylations
| PRJNA730412 | ENA
Project description:2’-O-methylation (Nm) is one of the most abundant RNA epigenetic modification and plays vital roles in the post-transcriptional regulation of gene expression. Current Nm mapping approaches are normally limited to highly abundant RNAs and have significant technical hurdles in mRNAs or relatively rare non-coding RNAs (ncRNAs). Here, we developed a new method for enriching Nm sites by using RNA exoribonuclease (M. genitalium RNase R, MgR) and periodate oxidation reactivity to eliminate 2’-hydroxylated (2’-OH) nucleosides, coupled with sequencing (Nm-REP-seq). We revealed several novel classes of Nm-containing ncRNAs as well as mRNAs in humans, mice, and drosophila. We found that some novel Nm sites are present at fixed positions in different tRNAs and are potential substrates of fibrillarin (FBL) methyltransferase mediated by snoRNAs. Importantly, we discovered, for the first time, that Nm located at the 3’-end of various types of ncRNAs and fragments derived from them. Our approach precisely redefines the genome-wide distribution of Nm and provides new technologies for functional studies of Nm-mediated gene regulation.
2021-03-25 | GSE157930 | GEO
Project description:Pseudouridine (Ψ) is an abundant RNA modification that is present in and affects the functions of diverse non-coding RNA species, including rRNA, tRNA and small nuclear RNA. Ψ also exists in mammalian mRNA and probably exhibits functional roles; however, functional investigations of mRNA Ψ modifications in mammals have been hampered by the lack of a quantitative method that detects Ψ at base precision. We have recently developed bisulfite-induced deletion sequencing (BID-seq), which provides the community with a quantitative method to map RNA Ψ distribution transcriptome-wide at single-base resolution. Here, we describe an optimized BID-seq protocol for mapping Ψ distribution across cellular mRNAs, which includes fast steps in both library preparation and data analysis. This protocol generates highly reproducible results by inducing high deletion ratios at Ψ modification within diverse sequence contexts, and meanwhile displayed almost zero background deletions at unmodified uridines. When used for transcriptome-wide Ψ profiling in mouse embryonic stem cells, the current protocol uncovered 8,407 Ψ sites from as little as 10 ng of polyA+ RNA input. This optimized BID-seq workflow takes 5 days to complete and includes four main sections: RNA preparation, library construction, next-generation sequencing (NGS) and data analysis. Library construction can be completed by researchers who have basic knowledge and skills in molecular biology and genetics. In addition to the experimental protocol, we provide BID-pipe ( https://github.com/y9c/pseudoU-BIDseq ), a user-friendly data analysis pipeline for Ψ site detection and modification stoichiometry quantification, requiring only basic bioinformatic and computational skills to uncover Ψ signatures from BID-seq data.
| S-EPMC11007761 | biostudies-literature
Project description:The ribose of RNA nucleotides can be 2'-O-methylated (Nm). Despite advances in high-throughput detection, the inert chemical nature of Nm still limits sensitivity and precludes mapping in mRNA. We leveraged the differential reactivity of 2'-O-methylated and 2'-hydroxylated nucleosides to periodate oxidation to develop Nm-seq, a sensitive method for transcriptome-wide mapping of Nm with base precision. Nm-seq uncovered thousands of Nm sites in human mRNA with features suggesting functional roles.
| S-EPMC5712428 | biostudies-literature
Project description:Transcriptome-Wide 2’-O-Methylation Mapping at Single-base Resolution by Nm-REP-seq
| PRJNA663360 | ENA
Project description:N4-acetylcytidine (ac4C) is an mRNA modification catalyzed by the enzyme N-acetyltransferase 10 (NAT10), with position-dependent effects on mRNA translation. This protocol details a procedure to map ac4C at base resolution using NaBH4-induced reduction of ac4C and conversion to thymidine followed by sequencing (RedaC:T-seq). Total RNA is ribodepleted and then treated with NaBH4 to reduce ac4C to tetrahydro-ac4C, which specifically alters base pairing during cDNA synthesis, allowing the detection of ac4C at positions called as thymidine following Illumina sequencing. For complete details on the use and execution of this protocol, please refer to Arango et al. (2022).1.
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Project description:[This corrects the article PMC5712428.].
| S-EPMC6994179 | biostudies-literature
Project description:3-Methylcytidine (m3C), a prevalent modification of transfer RNAs (tRNAs), was recently identified in eukaryotic messenger RNAs (mRNAs). However, its precise distribution and formation mechanisms in mRNAs remain elusive. Here, we develop a novel approach, m3C immunoprecipitation and sequencing (m3C-IP-seq), utilizing antibody enrichment to profile the m3C methylome at single-nucleotide resolution. m3C-IP-seq captures 12 cytoplasmic tRNA isoacceptors and 2 mitochondrial tRNA isoacceptors containing m3C modifications. Moreover, m3C-IP-seq permits the comprehensive profiling of m3C sites in mRNAs and long noncoding RNAs, with their presence reliant on a nuclear isoform of METTL8. A significant proportion of m3C sites is concentrated in the 3' untranslated region (3' UTR) of mRNAs and is associated with mRNA degradation. Additionally, m3C methylation is dynamic and responds to hypoxia. Collectively, our data demonstrate the widespread presence of m3C modification in the human transcriptome and provide a resource for functional studies of m3C-mediated RNA metabolism.
| S-EPMC11897884 | biostudies-literature
Project description:Functional characterization of pseudouridine (Ψ) in mammalian mRNA has been hampered by the lack of a quantitative method that maps Ψ in the whole transcriptome. We report bisulfite-induced deletion sequencing (BID-seq), which uses a bisulfite-mediated reaction to convert pseudouridine stoichiometrically into deletion upon reverse transcription without cytosine deamination. BID-seq enables detection of abundant Ψ sites with stoichiometry information in several human cell lines and 12 different mouse tissues using 10-20 ng input RNA. We uncover consensus sequences for Ψ in mammalian mRNA and assign different 'writer' proteins to individual Ψ deposition. Our results reveal a transcript stabilization role of Ψ sites installed by TRUB1 in human cancer cells. We also detect the presence of Ψ within stop codons of mammalian mRNA and confirm the role of Ψ in promoting stop codon readthrough in vivo. BID-seq will enable future investigations of the roles of Ψ in diverse biological processes.
| S-EPMC10017504 | biostudies-literature