Nm-seq maps 2'-O-methylation sites in human mRNA with base precision - PubMed (original) (raw)

Nm-seq maps 2'-O-methylation sites in human mRNA with base precision

Qing Dai et al. Nat Methods. 2017 Jul.

Erratum in

Abstract

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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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1

Nm-seq, a method based on oxidative cleavage for mapping 2′-_O_-methylation with base precision. (a) Schematic illustration. Fragmented RNA (I) is subjected to iterative oxidation–elimination–dephosphorylation (OED) cycles that remove 2′-hydroxylated nucleotides in the 3′-to-5′ direction to expose internal Nm sites at 3′ ends of fragments (II). Fragments ending with 2′-hydroxyl are then blocked by an incomplete cycle (OE, III), followed by adaptor ligation and library construction (IV). Paired-end sequencing (V) produces an asymmetric coverage profile, the uniform 3′ end of which corresponds to an Nm site shown in red below (G). Red triangles, Nm sites; n and gray shadow, number of OED cycles equals number of removed nucleotides, except when Nm is encountered; x, 3′-monophosphates representing blocked ends; blue and orange lines, 5′ and 3′ adaptors, respectively. (b) Chemical structures of 2′-_O_-methylated and 2′-hydroxylated RNA polymers at intermediate steps (I, II, III) of the method. (c) MALDI-TOF spectra of model RNA oligonucleotides, 3′-modified (red) and unmodified (blue), produced at intermediate steps listed on the right. AU, arbitrary units. (d) OE effectively blocks 3′ adaptor ligation to 2′-hydroxylated but not to 2′-_O_-methylated 3′ ends. N, RNA model ending with 2′-hydroxyl; Nm, RNA model ending with 2′-_O_-methyl; Np, RNA model ending with 3′ monophosphate.

Figure 2

Figure 2

Nm sites in HeLa mRNA. (a) LC-MS/MS quantification of Nm in total and rRNA-depleted RNA. The level of each modified nucleoside is presented as a percentage of the unmodified one. Mean values ± s.e.m. are shown; n = 3. (b) Typical Nm-seq plots of methylated transcripts. 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. (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 33% of all Nm sites. It appears on average immediately downstream to the methylated position. (e) Distribution of Nm sites among different amino acid codons.

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