Quantifying the RNA cap epitranscriptome reveals novel caps in cellular and viral RNA

Abstract

Chemical modification of transcripts with 5' caps occurs in all organisms. Here, we report a systems-level mass spectrometry-based technique, CapQuant, for quantitative analysis of an organism's cap epitranscriptome. The method was piloted with 21 canonical caps-m7GpppN, m7GpppNm, GpppN, GpppNm, and m2,2,7GpppG-and 5 'metabolite' caps-NAD, FAD, UDP-Glc, UDP-GlcNAc, and dpCoA. Applying CapQuant to RNA from purified dengue virus, Escherichia coli, yeast, mouse tissues, and human cells, we discovered new cap structures in humans and mice (FAD, UDP-Glc, UDP-GlcNAc, and m7Gpppm6A), cell- and tissue-specific variations in cap methylation, and high proportions of caps lacking 2'-O-methylation (m7Gpppm6A in mammals, m7GpppA in dengue virus). While substantial Dimroth-induced loss of m1A and m1Am arose with specific RNA processing conditions, human lymphoblast cells showed no detectable m1A or m1Am in caps. CapQuant accurately captured the preference for purine nucleotides at eukaryotic transcription start sites and the correlation between metabolite levels and metabolite caps.

Figure 1.

Analysis of 5′ cap structures in RNA by CapQuant. (A) Chemical structures of 5′ caps. (B) Workflow for CapQuant applied to eukaryotic mRNA. (C) A representative HPLC trace for the separation of the enzymatic digestion mixture of RNA. (D, E) Illustration of CapQuant for m⁷GpppAm in mRNA from mouse kidney (D), and NAD in total RNA from E. coli (E), showing HPLC elution profiles and MS/MS transitions (m/z X→Y) for unlabeled pure standard (top), the RNA sample (middle), and isotope-labeled standard spiked into the RNA sample (bottom). Similar illustrations of CapQuant for all other caps are shown in Supplementary Figure S8.

Figure 2.

Quantification of 5′ cap structures in cellular RNA and viral RNA genome by CapQuant. (A) mRNA from Human CCRF-SB cells. (B) mRNA from mouse C57BL/6 liver and kidney tissues. * P < 0.05, ** P < 0.01, two-tailed paired Student's t test. (C) mRNA from Saccharomyces cerevisiae W1588–4C cells. Exposure to hydrogen peroxide (H₂O₂) or methyl methanesulfonate (MMS) induces changes to the profile of 5′ cap structures in mRNA from Saccharomyces cerevisiae. From left to right: untreated, H₂O₂-treated, MMS-treated. ** P < 0.01, two-tailed unpaired Student's t test. (D) E. coli DH5α total RNA. (E) DENV-2 virus RNA genome. Values represent mean ± SD for three independent cultures for CCRF-SB, W1588–4C and DH5α, for three biological replicates of three mice and H₂O₂- or MMS-treated W1588–4C cells, and for three technical replicates of a single culture for DENV-2.

Figure 3.

Cap profile correlation with CAGE-analyzed transcription start site (TSS) nucleotide distribution. The frequency of A, G, C and T as the second nucleotide in m⁷GpppN caps was plotted against the distribution of these nucleotides at TSSs in (A) human (FANTOM5-weighted TSS), (B) mouse liver and kidney (FANTOM5-weighted TSS) and (C) Saccharomyces cerevisiae (YeasTSS-weighted TSS). TSS values were calculated as described in MATERIALS AND METHODS.