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.

CAP-MAP: Cap Analysis Protocol with Minimal Analyte Processing, a rapid and sensitive approach to analysing mRNA cap structures

“…Recently an alternative LC-MS method for detecting mRNA caps was reported [53]. In addition to dinucleotide caps, a variety of rare non-canonical metabolite mRNA caps were detected in mammalian cells including NAD-caps, which have only recently been described in mammalian cells and FAD-caps, UDP-Gluc-caps and UDP-GlucNAc-caps which were not previously known to exist in mammals [54].…”

The identity and methylation status of the first transcribed nucleotide in eukaryotic mRNA 5’ cap modulates protein expression in living cells

“…The proportion of A to m 6 A vary between studied cell lines, but usually is more than 50% (4–7). Numerous studies revealed that the 5’ cap composition is dependent on the organism, tissue or cell type (5,8,9).…”

“…The 5’ cap structures and cap proximal RNA sequences have been initially investigated by radioactivity-based chemo-enzymatic methods (1,2,4,9), but more recently, transcriptome-wide MS-based techniques enabled more robust and quantitative analyses leading to interesting discoveries (5). 2’- O Methylation is the most common modification of the first transcribed nucleotide in human cells and other vertebrates (1,2,5).…”

“…The 5’ cap structures and cap proximal RNA sequences have been initially investigated by radioactivity-based chemo-enzymatic methods (1,2,4,9), but more recently, transcriptome-wide MS-based techniques enabled more robust and quantitative analyses leading to interesting discoveries (5). 2’- O Methylation is the most common modification of the first transcribed nucleotide in human cells and other vertebrates (1,2,5). In mammalian cells 5’ terminal m 6 A occurs frequently (4) and interestingly, in contrary to adenine, m 6 A was observed also in significant amount as a 2’- O unmethylated species; in human B lymphoblasts m 6 A was as frequent as m 6 A m , whereas in mouse liver tissue the m 6 A m is 10 times more frequent (5).…”

Potential G-quadruplex forming sequences andN6-methyladenosine colocalize at human pre-mRNA intron splice sites

“…6 Processing of the 5′ and 3′ ends of mRNA has long been studied. 7,8 In contrast, our understanding of writing epitranscriptomic modifications and alternative mRNA splicing are rapidly advancing as a result of next-generation sequencing (NGS) and expansion of bioinformatic tools. [1][2][3][4] The best studied epitranscriptomic modification in mRNA is N 6 -methyladenosine (m 6 A; Figure 1A), in which deposition of the methyl group on A nucleotides occurs in specific sequence motifs; however, not all such sequences are modified.…”