A systems-level mass spectrometry-based technique for accurate and sensitive quantification of the RNA cap epitranscriptome

Abstract

Chemical modifications of transcripts with a 5' cap occur in all organisms and function in many aspects of RNA metabolism. To facilitate analysis of RNA caps, we developed a systems-level mass spectrometry-based technique, CapQuant, for accurate and sensitive quantification of the cap epitranscriptome. The protocol includes the addition of stable isotope-labeled cap nucleotides (CNs) to RNA, enzymatic hydrolysis of endogenous RNA to release CNs, and off-line enrichment of CNs by ion-pairing high-pressure liquid chromatography, followed by a 17 min chromatography-coupled tandem quadrupole mass spectrometry run for the identification and quantification of individual CNs. The total time required for the protocol can be up to 7 d. In this approach, 26 CNs can be quantified in eukaryotic poly(A)-tailed RNA, bacterial total RNA and viral RNA. This protocol can be modified to analyze other types of RNA and RNA from in vitro sources. CapQuant stands out from other methods in terms of superior specificity, sensitivity and accuracy, and it is not limited to individual caps nor does it require radiolabeling. Thanks to its unique capability of accurately and sensitively quantifying RNA caps on a systems level, CapQuant can reveal both the RNA cap landscape and the transcription start site distribution of capped RNA in a broad range of settings.

Fig. 1 ∣. Representative chemical structures of RNA caps described in this protocol.

(A) The canonical RNA caps following the scheme of (m)GpppN(m), with cap0 (m⁷GpppN), cap1 (m⁷GpppNm), m⁷Gpppm⁶Am and m^2,2,7GpppG depicted. Methylation sites are highligthed as Me in red font. (B) Metabolite caps following the scheme of XppN, with the oxidized form of NAD⁺ being shown. N refers to any nucleotide, X being any metabolite moiety, and R refers to side chains. NAD, nicotinamide adenine dinucleotide; dpCoA, dephospho coenzyme A; FAD, flavin adenine dinucleotide; UDP-Glc(NAc), uridine diphosphate–glucose (UDP-Glc) or N-acetylglucosamine (UDP-GlcNAc) which is distinguished in grey font.

Fig. 2 ∣. Workflow for the quantitative analysis of 5’ cap structures in cellular and tissue mRNA.

Stable isotope-labeled cap nucleotides (SIL-CNs) are denoted by the pink star to differentiate from endogenous CNs released during hydrolysis of mRNA by nuclease P1. The typical fragmentations of CNs are shown as dashed lines in the diagram in the inset panel showing the LC-MS/MS steps. The figure was created using Biorender (permission granted).

Fig. 3 ∣. Off-line HPLC chromatogram of an enzymatic digestion mixture of RNA.

Black solid lines drawn above or below the chromatogram peaks denotes the retention time range in which the fractions are collected, with the corresponding CNs in these fractions as labelled. Isobaric caps are coloured in violet (m⁷GpppG/GpppGm), yellow (m⁷GpppU/GpppUm), blue (m⁷GpppC/GpppCm), green (m⁷GpppA/GpppAm), and red (m⁷Gpppm⁶A/Gpppm⁶Am) to highlight their separation. This figure was originally published in Figure 1C of Wang et al. (2019) paper .

Fig. 4 ∣. Selected ion chromatograms of the MRM transitions used in the identification and quantification of the cap nucleotides (Table 4) are illustrated for two cap nucleotides in two different samples.

(A) m⁷GpppAm (top row) in mouse kidney mRNA, ¹⁵N₅-m⁷GpppAm standard (middle row), with a single MRM transition for m/z 806→136 (black) which is assigned to the product ion [Adenine + H]⁺ (bottom row). (B) NAD (top row) in E. coli total RNA with a zoom-in panel to illustrate the MRM transitions for m/z 664→136 (black), m/z 664→232 (red), and m/z 664→428 (blue), which correspond to [Adenine + H]⁺, [Adenosine − 2H₂O]⁺, and [ADP + H]⁺ (bottom row) respectively. A single MRM transition for m/z 664→136 (green) is used to monitor the ¹³C₅-NAD standard (middle row). The fragmentation of the CN structures is shown in the bottom row as black dashed arrows and the numbers are the mass-to-charge ratios (m/z) of the fragment ions. This figure was adapted from Figure 1D-E of Wang et al. (2019) paper .

Fig. 5 ∣. Quality control analysis of total RNA on an Agilent Bioanalyzer.

Total human CCRF-SB cell RNA (red trace) was resolved on an RNA Nano Chip in an Agilent 2100 Bioanalyzer. Size markers are noted in blue.

Fig. 6 ∣. Quality control analysis of poly(A)-tailed RNA on an Agilent Bioanalyzer.

Human CCRF-SB cell poly(A)-tailed RNA (red trace) was resolved on an RNA Pico Chip in an Agilent 2100 Bioanalyzer. Size markers are noted in blue.

Fig. 7 ∣. Analysis of LC-MS data for cap nucleotides in mRNA from human CCRF-SB cells.

This figure was adapted from Figure 2A and Table 1 in Wang et al. (2019). Bar graph values represent mean ± SD for three independent cultures for CCRF-SB, plotted using Graphpad Prism with 3 segments to the Y-axis representing the dynamic abundance in fmol/ug RNA per cap nucleotide as labelled in the X-axis.

Fig. 8 ∣. Transcriptional Start Site (TSS) cross-validation workflow using Galaxy server.

It begins with the import of available sequencing datasets from the cap-analysis gene expression (CAGE) approach to the Galaxy open-source platform for data analysis as outlined in the figure. The workflow can be accessed via https://usegalaxy.org/histories/list_published?f-username=alvin_chew.