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 (mC) formation in mammalian RNAs is still poorly understood. However, the recent discovery of as the second gene responsible for mC presence in RNA in fission yeast raises the possibility that multiple enzymes are involved in mC formation in mammals as well. Here, we report the discovery and characterization of three distinct mC-contributing enzymes in mice and humans. We found that methyltransferase-like (METTL) 2 and 6 contribute mC in specific tRNAs and that METTL8 only contributes mC to mRNA. MS analysis revealed that there is an ∼30-40% and ∼10-15% reduction, respectively, in and null-mutant cells, of mC in total tRNA, and primer extension analysis located METTL2-modified mC at position 32 of tRNA isoacceptors and tRNA 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. , however, modified only mRNA, as determined by biochemical and genetic analyses in null-mutant mice and two human mutant cell lines. Our findings provide the first evidence of the existence of mC modification in mRNA, and the discovery of METTL8 as an mRNA mC writer enzyme opens the door to future studies of other mC epitranscriptomic reader and eraser functions.

RNA methylation in nuclear pre‐mRNA processing

“…It is possible that m 1 A, like in some tRNA, stabilizes the tertiary conformation of mascRNA and thus promotes its yet unknown function in the cytoplasm. The additional methylation modifications m 3 C and m 5 C, which are also typically found in ncRNA (such as tRNA and rRNA) were recently also reported in mRNA (Clark, Evans, Dominissini, Zheng, & Pan, 2016;Cozen et al, 2015;Iwanami & Brown, 1968a, 1968bSquires et al, 2012;Xu et al, 2017).…”

“…The additional methylation modifications m 3 C and m 5 C, which are also typically found in ncRNA (such as tRNA and rRNA) were recently also reported in mRNA (Clark, Evans, Dominissini, Zheng, & Pan, ; Cozen et al, ; Iwanami & Brown, , ; Squires et al, ; Xu et al, ).…”

A new modification for mammalian messenger RNA

“…Instead, Xu et al (2) provide definitive proof through stringent purification of mRNA and quantification by LC-MS/MS that METTL8 acts on mRNA, functionally annotating this enzyme and defining m 3 C as a modification in mRNA for the first time in mammalian systems. Specifically, tRNA was first removed from the total RNA sample by size-exclusion chromatography.…”

“…The studies of these modifications, particularly in the context of mRNA and lncRNA, embody the new concept of "epitranscriptomics," in which the functional significance of chemical alterations is controlled by three groups of proteins: "writers" to install, "erasers" to remove, and "readers" to recognize modifications and thus determine the cellular fate of the modified RNA species. Xu et al (2) now report the characterization of three "writers" in the form of mammalian methyltransferases anticipated to introduce N 3 -methylcytosine (m 3 C) 2 modifications. Their data show that two of the enzymes act on tRNA as suspected, whereas the third surprisingly uses mRNA as a substrate, defining a new modification for this RNA species in mammals.…”

“…E-mail: chuanhe@uchicago.edu. 2 The abbreviations used are: m The work by Fu and colleagues (6) provides several immediate angles to explore. Initial primer extension assays suggested METTL6 acts near position 32 of the two serine-specific tRNAs, but further data will be needed to determine the specific substrate nucleotide.…”