Guanosine 10 methylation by TRMT11-TRMT112 optimizes human mitochondrial tRNAs for efficient translation

Modified nucleotides are essential determinants of RNA function, and identifying the enzymes that install them is fundamental to understanding their cellular roles. Here, we show that the human RNA methyltransferase TRMT11 and its cofactor TRMT112 are imported into mitochondria via N-terminal targeting signals. Using a recently developed N2-methylguanosine (m2G)-sensitive DNAzyme, we demonstrate that TRMT11 catalytic activity and interaction with TRMT112 are required for installation of m2G at position 10 in 13 mitochondrial (mt-)tRNAs. The crosslinking profile of TRMT11 on mt-tRNAs experimentally supports a model of the TRMT11-TRMT112-mt-tRNATrp complex in which the THUMP domain contacts the 3' end of the acceptor stem, and G10 is flipped into the S-adenosylmethionine binding pocket for methylation. Transcriptome-wide mapping reveals that TRMT11 interacts with most nuclear-encoded and mt-tRNAs, but only methylates a subset. In vitro reconstitution of TRMT11-TRM112-mediated methylation defines key structural requirements for m2G10 installation across different mt-tRNAs, and reveals how pathogenic mutations influence this modification. TRMT11-TRMT112 recognizes folded mt-tRNAs, and in the degenerate mt-tRNALys, m1A9 strongly enhances m2G10 methylation efficiency. Loss of m2G10 modifications alters the conformation of numerous mt-tRNAs, perturbs mitochondrial protein synthesis, and impairs oxidative phosphorylation, highlighting an essential role of this modification in maintaining mitochondrial function.