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Nm-Mut-seq: The base-resolution quantitative method for mapping transcriptome-wide 2’-O-methylations

ABSTRACT: Nm-Mut-seq: The base-resolution quantitative method for mapping transcriptome-wide 2’-O-methylations

PROVIDER: PRJNA730412 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR14562311.fastq.gz	Fastqsanger.gz
	SRR14562312.fastq.gz	Fastqsanger.gz
	SRR14562313.fastq.gz	Fastqsanger.gz
	SRR14562314.fastq.gz	Fastqsanger.gz
	SRR14562315.fastq.gz	Fastqsanger.gz

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Nm-Mut-seq: The base-resolution quantitative method for mapping transcriptome-wide 2’-O-methylations

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.

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Nm-seq finds thousands of modified 2’-O-methylation sites in mRNA with base precision

Project description:Nm-seq maps 2'-O-methylation sites in human mRNA with base precision 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.

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Nm-Mut-seq: a base-resolution quantitative method for mapping transcriptome-wide 2'-O-methylation.

Project description: Not available

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Transcriptome-Wide 2’-O-Methylation Mapping at Single-base Resolution by Nm-REP-seq

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.

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Nm-REP-seq Reveals 2’-O-Methylation Landscape at the 3'-end of Noncoding RNAs with Single-base Resolution

Project description:Many non-coding RNAs (ncRNAs), such as piRNAs, miRNAs and siRNAs, contain Nm at the 3'-end nucleotides, protecting the 3’-ends form terminal nucleotide addition or exonucleolytic degradation, but whether other kinds of RNAs contain a 3'-terminal Nm remain unknown. Here, we developed Nm-REP-seq to globally profile the 3'end-Nm sites and we revealed several novel classes of Nm-containing ncRNAs in mice and drosophila. Importantly, we discovered that Nm distribute at the end of miRNA, snoRNA, snRNA, tRNA as well as fragments derived from them. Collectively, our approach precisely redefines the genome-wide distribution of Nm and provides new technologies for functional studies of Nm-mediated gene regulation. Method: We developed Nm-REP-seq to globally profile the 3'-end Nm sites. Results: We discovered that Nm distribute at the end of miRNA, snoRNA, snRNA, tRNA as well as fragments derived from them. Conclusion:Collectively, our approach precisely redefines the genome-wide distribution of Nm and provides new technologies for functional studies of Nm-mediated gene regulation.

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