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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This work was supported by the US National Institutes of Health NHGRI RM1 HG008935 to C.H.; a grant from the Kahn Family Foundation to D.D. and G.R.; and grants from the Ernest and Bonnie Beutler Research Program, Flight Attendant Medical Research Institute (FAMRI) and the Israeli Centers of Excellence (I-CORE) Program (ISF grants no. 41/11 and no. 1796/12) to G.R. C.H. is an investigator of the Howard Hughes Medical Institute (HHMI). G.R. is a member of the Sagol Neuroscience Network and holds the Djerassi Chair for Oncology at the Sackler Faculty of Medicine, Tel-Aviv University, Israel. D.D. was supported by a Human Frontier Science Program (HFSP) long-term fellowship. Q.D. is supported by the National Institutes of Health grant K01 HG006699. We wish to thank M. Salmon-Divon for advice and help with bioinformatic analysis and R. Mashiach for help with chemical structure drawings.
The authors declare no competing financial interests.
Integrated supplementary information
(a-b), Nm-seq profiles of human 18S (a) and 28S (b) rRNA above MCC-determined optimal threshold (blue). Known Nm sites are shown as red bars below. (c-d), Receiver Operating Characteristic (ROC, orange) and Mathews Correlation Coefficient (MCC, green) curves for human 18S (c) and 28S (d) rRNA plotted using increasing normalized 3′ end coverage thresholds at each position.
Nm-seq plots of methylated transcripts: (a) NKIRAS1 (b) KLHL5. Normalized summed sequence coverage of Nm-seq and input are shown below and above the transcript, respectively. Individual paired-end reads within the Nm site window are shown in magnification.
(a) Distribution of 2′-O-methyl sites between the four nucleobases in the various transcript segments and overall. (b) Fraction of Nm sites detected within mRNA and ncRNA. (c) The percentage of methylated genes according to the number of Nm sites per gene. (d) The percentage of methylated genes increases with expression level.
Supplementary Figure 4 RNA secondary structure surrounding Nm sites, m6Am in mRNA and Gene Ontology (GO) analysis
The secondary structures of a 200-nt window centered on Nm sites was analyzed using the Structure Surfer tool based on: (a) PARS score (b) ds/ssRNA score and (c) DMS-seq. (d) LC-MS/MS quantification of internal (i.e., excluding the first transcribed nucleotide) m6A and internal m6Am in HeLa mRNA. The level of each modified nucleoside is presented as a percentage of the unmodified one. Mean values ± s.e.m. are shown, n = 3. (e) GO analysis of Nm-methylated HeLa genes relative to all adequately expressed genes (above the 1st quartile) reveals enrichment of GO terms related to cell-cell interactions, splicing and more (fold enrichment ≥ 2, Bonferroni corrected P ≤ 0.005). Fold-enrichment and P values are indicated for each category.
(a) Distribution of Nm sites between exons, introns and alternatively spliced regions. (b) Metagene profile of Nm site distribution along a normalized mRNA transcript. (c) Metagene profile of Nm sites distribution relative to the first and nearest splice sites in a 400-nt non-normalized window. (d) Metagene profile of Nm site distribution along a normalized intron.
(a) Distribution of 2′-O-methyl sites between the four nucleobases in the various transcript segments and overall. (b) Fraction of Nm sites detected within mRNA and ncRNA. (c) Metagene profile of Nm sites distribution along a normalized mRNA transcript illustrated below. (d) Sequence logo of the most enriched motif identified by HOMER in 58.7% of all HEK293 Nm sites. (e) The percentage of methylated genes according to the number of Nm sites per gene.
(a) HEK293 Nm sites in different transcript segments of coding genes. (b) Distribution of Nm sites between exons, introns and alternatively spliced regions. (c) Metagene profile of Nm sites distribution relative to the first (blue) and nearest (red) splice sites in a 400-nt non-normalized window. (d) Distribution of Nm sites between the three codon positions. (e) Distribution of Nm sites among different amino acid codons.
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Dai, Q., Moshitch-Moshkovitz, S., Han, D. et al. Nm-seq maps 2′-O-methylation sites in human mRNA with base precision. Nat Methods 14, 695–698 (2017). https://doi.org/10.1038/nmeth.4294
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