METTL8 is required for 3-methylcytosine modification in human mitochondrial tRNAs

A subset of eukaryotic tRNAs is methylated in the anticodon loop to form the 3-methylcytosine (m3C) modification. In mammals, the number of tRNAs containing m3C has expanded to include mitochondrial (mt) tRNA-Ser-UGA and mt-tRNA-Thr-UGU. Whereas the enzymes catalyzing m3C formation in nuclear- encoded cytoplasmic tRNAs have been identified, the proteins responsible for m3C modification in mt- tRNAs are unknown. Here, we show that m3C formation in human mt-tRNAs is dependent upon the Methyltransferase-Like 8 (METTL8) enzyme. We find that METTL8 is a mitochondria-associated protein that interacts with mitochondrial seryl-tRNA synthetase along with mt-tRNAs containing m3C. Human cells deficient in METTL8 exhibit loss of m3C modification in mt-tRNAs but not nuclear-encoded tRNAs. Consistent with the mitochondrial import of METTL8, the formation of m3C in METTL8-deficient cells can be rescued by re-expression of wildtype METTL8 but not by a METTL8 variant lacking the N-terminal mitochondrial localization signal. Notably, METTL8-deficiency in human cells causes alterations in the native migration pattern of mt-tRNA-Ser-UGA suggesting a role for m3C in tRNA folding. Altogether, these findings demonstrate that METTL8 is required for m3C formation in mitochondrial tRNAs and uncover a potential role for m3C modification in mitochondrial tRNA structure.

The methyltransferase TrmA facilitates tRNA folding through interaction with its RNA-binding domain

tRNAs are the most highly modified RNAs in all cells, and formation of 5-methyluridine (m5U) at position 54 in the T arm is a common RNA modification found in all tRNAs. The m5U modification is generated by the methyltransferase TrmA. Here, we test and prove the hypothesis that Escherichia coli TrmA has dual functions, acting both as a methyltransferase and as a tRNA chaperone. We identify two conserved residues, F106 and H125, in the RNA-binding domain of TrmA, which interact with the tRNA elbow and are critical for tRNA binding. Co-culture competition assays reveal that the catalytic activity of TrmA is important for cellular fitness, and that substitutions of F106 or H125 impair cellular fitness. We directly show that TrmA enhances tRNA folding in vitro independent of its catalytic activity. In conclusion, our study suggests that F106 and H125 in the RNA-binding domain of TrmA act as a wedge disrupting tertiary interactions between tRNA’s D arm and T arm; this tRNA unfolding is the mechanistic basis for TrmA’s tRNA chaperone activity. TrmA is the second tRNA modifying enzyme next to the pseudouridine synthase TruB shown to act as a tRNA chaperone supporting a functional link between RNA modification and folding.

Single-nucleotide control of tRNA folding cooperativity under near-cellular conditions

RNA folding is often studied by renaturing full-length RNA in vitro and tracking folding transitions. However, the intracellular transcript folds as it emerges from the RNA polymerase. Here, we investigate the folding pathways and stability of numerous late-transcriptional intermediates of yeast and Escherichia coli transfer RNAs (tRNAs). Transfer RNA is a highly regulated functional RNA that undergoes multiple steps of posttranscriptional processing and is found in very different lengths during its lifetime in the cell. The precursor transcript is extended on both the 5′ and 3′ ends of the cloverleaf core, and these extensions get trimmed before addition of the 3′-CCA and aminoacylation. We studied the thermodynamics and structures of the precursor tRNA and of late-transcriptional intermediates of the cloverleaf structure. We examined RNA folding at both the secondary and tertiary structural levels using multiple biochemical and biophysical approaches. Our findings suggest that perhaps nature has selected for a single-base addition to control folding to the functional 3D structure. In near-cellular conditions, yeast tRNAPhe and E. coli tRNAAla transcripts fold in a single, cooperative transition only when nearly all of the nucleotides in the cloverleaf are transcribed by indirectly enhancing folding cooperativity. Furthermore, native extensions on the 5′ and 3′ ends do not interfere with cooperative core folding. This highly controlled cooperative folding has implications for recognition of tRNA by processing and modification enzymes and quality control of tRNA in cells.

RNA modification enzyme TruB is a tRNA chaperone

Cellular RNAs are chemically modified by many RNA modification enzymes; however, often the functions of modifications remain unclear, such as for pseudouridine formation in the tRNA TΨC arm by the bacterial tRNA pseudouridine synthase TruB. Here we test the hypothesis that RNA modification enzymes also act as RNA chaperones. Using TruB as a model, we demonstrate that TruB folds tRNA independent of its catalytic activity, thus increasing the fraction of tRNA that can be aminoacylated. By rapid kinetic stopped-flow analysis, we identified the molecular mechanism of TruB’s RNA chaperone activity: TruB binds and unfolds both misfolded and folded tRNAs thereby providing misfolded tRNAs a second chance at folding. Previously, it has been shown that a catalytically inactive TruB variant has no phenotype when expressed in an Escherichia coli truB KO strain [Gutgsell N, et al. (2000) RNA 6(12):1870–1881]. However, here we uncover that E. coli strains expressing a TruB variant impaired in tRNA binding and in in vitro tRNA folding cannot compete with WT E. coli. Consequently, the tRNA chaperone activity of TruB is critical for bacterial fitness. In conclusion, we prove the tRNA chaperone activity of the pseudouridine synthase TruB, reveal its molecular mechanism, and demonstrate its importance for cellular fitness. We discuss the likelihood that other RNA modification enzymes are also RNA chaperones.