Three distinct 3-methylcytidine (m3C) methyltransferases modify tRNA and mRNA in mice and humans

Abstract

Chemical RNA modifications are central features of epitranscriptomics, highlighted by the discovery of modified ribonucleosides in mRNA and exemplified by the critical roles of RNA modifications in normal physiology and disease. Despite a resurgent interest in these modifications, the biochemistry of 3-methylcytidine (m³C) formation in mammalian RNAs is still poorly understood. However, the recent discovery of trm141 as the second gene responsible for m³C presence in RNA in fission yeast raises the possibility that multiple enzymes are involved in m³C formation in mammals as well. Here, we report the discovery and characterization of three distinct m³C-contributing enzymes in mice and humans. We found that methyltransferase-like (METTL) 2 and 6 contribute m³C in specific tRNAs and that METTL8 only contributes m³C to mRNA. MS analysis revealed that there is an ∼30-40% and ∼10-15% reduction, respectively, in METTL2 and -6 null-mutant cells, of m³C in total tRNA, and primer extension analysis located METTL2-modified m³C at position 32 of tRNA^Thr isoacceptors and tRNA^Arg(CCU) We also noted that METTL6 interacts with seryl-tRNA synthetase in an RNA-dependent manner, suggesting a role for METTL6 in modifying serine tRNA isoacceptors. METTL8, however, modified only mRNA, as determined by biochemical and genetic analyses in Mettl8 null-mutant mice and two human METTL8 mutant cell lines. Our findings provide the first evidence of the existence of m³C modification in mRNA, and the discovery of METTL8 as an mRNA m³C writer enzyme opens the door to future studies of other m³C epitranscriptomic reader and eraser functions.

Keywords: Mettl2; Mettl6; Mettl8; RNA methyltransferase; human; m3C; mRNA; mice; mouse; transfer RNA (tRNA).

Figure 1.

Contribution of METTL2 and METTL6 to tRNA m³C in mouse and human cell lines. A, structure of m³C. Positions 2–4 are involved in Watson-Crick base pairing, with the N³-methylation serving as a block to polymerases. This polymerase-blocking phenomenon was exploited to map the location of methylations in tRNA. B and C, relative m³C levels in tRNA isolated liver and brain tissues, respectively. Samples from wild-type (WT), Mettl2 KO (M2 KO), Mettl6 mutant (M6 KO), and Mettl8 KO (M8 KO) mice were analyzed. D, relative m³C levels in HEK293T wild-type and METTL2 KO cell lines (loss of both METTL2A and -2B). E, relative m³C levels in HCT116 METTL8 wild-type and knock-out sample. m³C modification levels were normalized against levels of canonical cytidine. Data represent mean ± S.D. for at least three biological replicates, with asterisks denoting significant differences by Student's t test; **, p < 0.01. ns, not significant.

Figure 2.

Mouse METTL2 is responsible for position 32 modification of tRNA^Thr(UGU) and tRNA^Arg(CCU). A and B, schematic showing one tRNA^Thr(UGU) and one tRNA^Arg(CCU) isoacceptor from mice, with red curved lines denoting the coverage of probes used in primer extension assay to map polymerase-blocking modification at position C32. C, primer extension assay using probes targeting tRNA^Thr(UGU) using RNA from wild type (WT), Mettl2 KO (M2 KO), and Mettl6 (M6 KO) mutants. Free probes are 22 nt long. D, primer extension for tRNA^Arg(CCU). Marker lane (M) contains a 56-nt-long ssDNA probe.

Figure 3.

RNA-dependent interaction between METTL6 and SARS. HEK293T cells were transfected with either FLAG-METTL6 (M6) or FLAG-METTL6-ΔSAM (M6Δ). Equal amounts (protein level) of cell extracts were immunoprecipitated (IP) with either nonspecific IgG as a control or FLAG antibody, and treated with either buffer (−), RNase (+), or DNase (+), followed by SDS-PAGE, blotting, and probing with SARS or FLAG antibodies. Blots with Input cell extracts were probed with SARS and β-actin antibodies.

Figure 4.

Poly(A) mRNA enrichment and m³C levels monitored during purification. A, schematics of poly(A) mRNA enrichment and separation of 28S and 18S rRNA. B, typical electropherogram for the following: 1. total RNA; 2. RNA >5.8S isolated by SEC; 3. poly(A) RNA enriched by oligo(dT) selection; 4. poly(A)-enriched mRNA after rRNA depletion; 5. 28S; and 6. 18S rRNA purified by SEC. C, LC-MS/MS quantitation of m³C level in tRNA, RNA larger than 5.8S rRNA, oligo(dT)-enriched mRNA, ribo-zero-treated mRNA, 18S rRNA, 28S rRNA. D, LC-MS/MS quantitation of m³C, m⁵C, m¹A, and m⁶A in human HCT116 mRNA purified by SEC, poly(A)-enrichment, and rRNA depletion. (The level of each modified ribonucleoside is expressed relative to its unmodified form. Data represent mean ± S.D. for n > = 3.)

Figure 5.

METTL8 catalyzes formation of m³C in mRNA in mouse liver tissues and human cell lines. mRNA was isolated from mouse liver tissue, human HCT116, and HeLa S3 cells using the purification scheme shown in Fig. 4A. A, LC-MS/MS chromatogram for m³C, m⁴C, and m⁵C in wild-type mice (WT) and mice lacking Mettl2 (M2 KO), Mettl6 (M6 KO), or Mettl8 (M8 KO), as well as a double knock-out for METTL6 and -8 (M6,8 KO). Peak areas are noted numerically above each peak. B, LC-MS/MS chromatogram for m³C, m⁴C, and m⁵C in HeLa S3 METTL8 KO cells transfected with a vector expressing either wild-type (M8(WT)) or a SAM-binding mutant METTL8 (M8(ΔSAM)). Peak areas are noted numerically above each peak. C–E, LC-MS/MS quantification of m³C levels in mouse liver tissue (C), HCT116 cells (D), and HeLa S3 cells (E). Abbreviations are the same as in A and B. (The level of m³C is expressed relative to canonical cytidine. Data with error bars represent mean ± S.D. for at least three biological replicates.)