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

Chen, Li; Zhang, Li-Sheng; Ye, Chang; Zhou, Huiqing; Liu, B.; Gao, Boyang; Yu, Yi; Zhao, Changming; He, Chuan; Dickinson, Bryan C.

doi:10.1038/s41422-023-00836-w

Cited by 8 publications

(13 citation statements)

References 15 publications

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“…Spike-in probes are not necessary for UBS-seq, since the chemical treatment displays an extremely high conversion ratio with no noticeable preference in sequence context around unmethylated cytidines. For other methods mentioned in this Account, such as BID-seq, N m -Mut-seq, and m 1 A-quant-seq, ,, calibration probes are recommended for reproducing observations on modification stoichiometry when using different batches of engineered or commercially available RT enzymes. These RT enzymes may have sequence preferences.…”

Section: Discussionmentioning

confidence: 99%

“…Indeed, a new HIV RT variant, RT-41B4, was selected to yield notable misincorporation signatures at N m -modified sites. In the presence of adjusted dATP/dNTP ratios during RT, we developed N m -Mut-seq (an RT-mutation-based N m mapping method) that maps C m , G m , and A m methylations at single-base resolution with stoichiometry information (Figure b). To validate N m -Mut-seq, we mapped almost all known C m , G m , and A m sites on human rRNAs with high misincorporation rates and without observing any false positives at unmodified sites or other rRNA modifications .…”

Section: Introductionmentioning

confidence: 99%

“…In the presence of adjusted dATP/dNTP ratios during RT, we developed N m -Mut-seq (an RT-mutation-based N m mapping method) that maps C m , G m , and A m methylations at single-base resolution with stoichiometry information (Figure b). To validate N m -Mut-seq, we mapped almost all known C m , G m , and A m sites on human rRNAs with high misincorporation rates and without observing any false positives at unmodified sites or other rRNA modifications . Applying N m -Mut-seq to HepG2 cellular mRNA, we uncovered around 1000 C m , G m , and A m sites, a subset of which were validated by multiple orthogonal methods, further confirming the fidelity of the method.…”

Section: Introductionmentioning

confidence: 99%

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Base-Resolution Sequencing Methods for Whole-Transcriptome Quantification of mRNA Modifications

Zhang,

Dai,

2023

Acc. Chem. Res.

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Metrics & MoreArticle Recommendations * sı Supporting Information CONSPECTUS: RNA molecules are not merely a combination of four bases of A, C, G, and U. Chemical modifications occur in almost all RNA species and play diverse roles in gene expression regulation. The abundant cellular RNAs, such as ribosomal RNA (rRNA) and transfer RNA (tRNA), are known to have the highest density of RNA modifications, which exert critical functions in rRNA and tRNA biogenesis, stability, and subsequent translation.In recent years, modifications on low-abundance RNA species in mammalian cells, such as messenger RNA (mRNA), regulatory noncoding RNA (ncRNA), and chromatin-associated RNA (caRNA), have been shown to contain multiple different chemical modifications with functional significance.As the most abundant mRNA modification in mammals, N 6methyladenosine (m 6 A) affects nearly every stage of mRNA processing and metabolism, with the antibody-based m 6 A-MeRIPseq (methylated RNA immunoprecipitation sequencing) followed by high-throughput sequencing widely employed in mapping m 6 A distribution transcriptome-wide in diverse biological systems. In addition to m 6 A, other chemical modifications such as pseudouridine (Ψ), 2′-O-methylation (N m ), 5-methylcytidine (m 5 C), internal N 7 -methylguanosine (m 7 G), N 1 -methyladenosine (m 1 A), N 4 -acetylcytidine (ac 4 C), etc. also exist in polyA-tailed RNA in mammalian cells, requiring effective mapping approaches for whole-transcriptome profiling of these non-m 6 A mRNA modifications. Like m 6 A, the antibody-based enrichment followed by sequencing has been the primary method to study distributions of these modifications. Methods to more quantitatively map these modifications would dramatically improve our understanding of distributions and modification density of these chemical marks on RNA, thereby bettering informing functional implications. In this Account, aimed at both single-base resolution and modification fraction quantification, we summarize our recent advances in developing a series of chemistry-or biochemistry-based methods to quantitatively map RNA modifications, including m 6 A, Ψ, m 5 C, m 1 A, 2′-O-methylation (N m ), and internal m 7 G, in mammalian mRNA at base resolution. These new methods, including m 6 A-SAC-seq, eTAM-seq, BID-seq, UBS-seq, DAMM-seq, m 1 A-quant-seq, Nm-Mut-seq, and m 7 G-quant-seq, promise to conduct base-resolution mapping of most major mRNA modifications with low RNA input and uncover dynamic changes in modification stoichiometry during biological and physiological processes, facilitating future investigations on these RNA modifications in regulating cellular gene expression and as potential biomarkers for clinical diagnosis and prognosis. These quantitative sequencing methods allow the mapping of most mRNA modifications with limited input sample requirements. The same modifications on diverse RNA species, such as caRNA, ncRNA, nuclear nascent RNA, mitochondrial RNA, cell-free RNA (cfRNA), etc., could be sequenced using the same methods.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Base-Resolution Sequencing Methods for Whole-Transcriptome Quantification of mRNA Modifications

Zhang,

Dai,

2023

Acc. Chem. Res.

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“…NJU-seq takes advantage of the resistance of Nm to cleavage by Mycoplasma genitalium RNase R (MgR) ( Tang et al 2024 ). Methods exploiting the steric properties of Nm include 2OMe-seq and MeTH-seq, which detect the termination of RT at Nm sites under restrictive conditions with either low deoxynucleotide triphosphate (dNTP) or low magnesium concentrations ( Incarnato et al 2017 ; Bartoli et al 2018 ), and Nm-Mut-seq, which uses an engineered reverse transcriptase to identify Nm sites as base mutations in the RT step, but is unable to detect Um ( Chen et al 2023 ). Emerging reports suggest that nanopore sequencing can map Nm at ±2 nt resolution based on Nm-induced changes in the current signal ( Stephenson et al 2022 ).…”

Section: Mapping and Measurement Of Nmmentioning

confidence: 99%

“…For Nm sites in rRNA, the stoichiometry of Nm determined by RiboMeth-seq correlates well with the Nm stoichiometry measured by liquid chromatography (LC) with tandem mass spectrometry (MS) ( Marchand et al 2016 ; Motorin and Marchand 2018 ; Taoka et al 2018 ). 2OMe-seq, Nm-Mut-seq, and NJU-seq can capture relative but not absolute Nm fraction with the use of spiked-in in vitro transcribed RNA controls ( Incarnato et al 2017 ; Chen et al 2023 ; Tang et al 2024 ). Low-throughput methods can also measure Nm fraction: RT-based methods such as RTL-P (RT at low dNTP concentrations followed by PCR) provide a semi-quantitative measure of Nm fraction ( Dong et al 2012b ; Elliott and Holley 2021 ), while Nm-VAQ (Nm validation and absolute quantification), which uses site-specific chimeric RNA/DNA probes to detect resistance to RNase H cleavage, is reported to allow absolute quantification of Nm stoichiometry ( Tang et al 2024 ).…”

Section: Mapping and Measurement Of Nmmentioning

confidence: 99%

2′-O-methylation (Nm) in RNA: progress, challenges, and future directions

Zhou,

Pecot,

Holley

2024

RNA

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RNA 2′-O-methylation (Nm) is highly abundant in noncoding RNAs including ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA), and occurs in the 5′ cap of virtually all messenger RNAs (mRNAs) in higher eukaryotes. More recently, Nm has also been reported to occur at internal sites in mRNA. High-throughput methods have been developed for the transcriptome-wide detection of Nm. However, these methods have mostly been applied to abundant RNAs such as rRNA, and the validity of the internal mRNA Nm sites detected with these approaches remains controversial. Nonetheless, Nm in both coding and noncoding RNAs have been demonstrated to impact cellular processes including translation and splicing. In addition, Nm modifications at the 5′ cap and possibly at internal sites in mRNA serve to prevent the binding of nucleic acid sensors, thus preventing the activation of the innate immune response by self mRNAs. Finally, Nm has been implicated in a variety of diseases including cancer, cardiovascular diseases, and neurologic syndromes. In this review, we discuss current challenges in determining the distribution, regulation, function, and disease relevance of Nm, as well as potential future directions for the field.

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Base-resolution quantitative DAMM-seq for mapping RNA methylations in tRNA and mitochondrial polycistronic RNA

Zhang,

Ju,

Jiang

et al. 2023

Enzymes in RNA Science and Biotechnology: Part B

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

Cited by 8 publications

References 15 publications

Base-Resolution Sequencing Methods for Whole-Transcriptome Quantification of mRNA Modifications

Base-Resolution Sequencing Methods for Whole-Transcriptome Quantification of mRNA Modifications

2′-O-methylation (Nm) in RNA: progress, challenges, and future directions

Base-resolution quantitative DAMM-seq for mapping RNA methylations in tRNA and mitochondrial polycistronic RNA

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