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 tRNAThr isoacceptors and tRNAArg(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.
Highlights
Chemical RNA modifications are central features of epitranscriptomics, highlighted by the discovery of modified ribonucleosides in messenger RNA (mRNA) and exemplified by the critical roles of RNA modifications in normal physiology and disease
Building on decades of study of transfer and ribosomal RNA, the resurgent interest in RNA modifications as central features of epitranscriptomics has been sparked by the discovery of modified ribonucleosides in messenger RNA [1, 2] and by the discovery of novel functions for non-coding RNA modifications in normal physiology and disease [3,4,5,6]
Using HPLC-coupled triple quadrupole mass spectrometry (LC-MS/ MS), we both identified and quantified m3C in tRNA fractions in brain and liver tissues from wild-type (WT) mice and Mettl2, -6, and -8 mutants. tRNA from both tissues of Mettl2 KO mice showed an ϳ35% reduction of m3C compared with WT tissues, whereas the Mettl6 mutants showed an ϳ12% m3C reduction (Fig. 1, B and C)
Summary
To define the catalytic activity of METTL2, -6, and -8 with tRNA, we generated null-mutant mice and cell lines by CRISPR/Cas. METTL6 bears homology with Trm141 in S. pombe, which targets serine tRNAs, so we assessed the presence of m3C32 in tRNASer(AGA) and tRNASer(GCU) using the primer extension assay This revealed the presence of read-through bands consistent with loss of RTblocking modification(s) near position 32 on tRNASer(AGA) and tRNASer(GCU) in Mettl KO cells (supplemental Fig. S8, C and D). Ectopic expression of METTL8 in HeLa S3 METTL8 KO cells for 3 days restores the m3C level in mRNA to ϳ59%, whereas complementation with a SAM-binding site mutant of METTL8 (METTL8-⌬SAM) failed to increase m3C (Fig. 5E) These results establish METTL8 as the m3C writer in mRNA in mouse liver tissue and two human cell lines. More work such as ribosome profiling is needed to reveal unrevealed biological functions of m3C
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