Fig 2: In vitro reconstitution of METTL8 methyltransferase activity on mt-tRNA-Thr-UGU. Purified METTL8 catalyzes m3C formation on mt-tRNA-Thr-UGU in vitro. A, representative gel of primer extension assay to monitor the presence of m3C in mt-tRNA-Thr-UGU after incubation with either control or purified METTL8. B, quantification of m3C formation in (A). Primer extension analysis was repeated three times and error bars represent the SD from the mean. m3C, 3-methylcytosine; METTL8, methyltransferase-like 8; mt, mitochondrial; RT, reverse transcriptase; >, labeled oligonucleotide used for primer extension.

Fig 3: METTL8 is required for efficient m3C formation in mitochondrial tRNAs in vivo.A, CRISPR/Cas9 gene KO strategy depicting sequence guide RNAs (sgRNAs) targeting exon 3 of the human METTL8 gene. B, genomic PCR demonstrating the loss of exon 3 in METTL8-KO cell lines. Deletion of exon 3 results in a 72 base-pair deletion. C, immunoblot analysis of METTL8 expression in WT versus METTL8-KO human cell lines. Actin was used as a loading control. D, (i) schematic of the Positive Hybridization in the Absence of Modification (PHA) to monitor m3C status. PHA probe spans position C32 while the control probe spans a region lacking modifications on the same tRNA. (ii) schematic of primer extension assay to monitor m3C modification. E, Northern blot analysis using the PHA assay with probes designed to detect m3C at position 32 and a control probe that hybridizes to a different area of the same tRNA. PHA quantification represents the ratio of PHA versus control probe signal expressed relative to the WT1 cell line. Northern blotting and quantification were performed on three independent cell preparations for each cell line with comparable results. F, representative gel from primer extension assays to monitor the presence of m3C in mt-tRNA-Thr-UGU from the indicated cell lines. G, quantification of m3C formation in (F). Primer extension analysis was repeated three times and bars represent the standard deviation from the mean. METTL8, methyltransferase-like 8; mt, mitochondrial; m1A, 1-methyladenosine; m3C, 3-methylcytosine; RT, reverse transcriptase; >, labeled oligonucleotide used for primer extension; .

Fig 4: Identification of METTL8-interacting proteins.A, silver stain analysis of Strep-Tactin purifications from human 293T cell lines expressing either vector control or METTL8-Strep. B, LC-MS analysis of peptides present in Strep-METTL8 purifications. Table lists a selection of proteins that were enriched in the Strep-METTL8 purification compared to empty vector control. C, schematic comparison of SARS1 versus SARS2 with aminoacyl synthetase domain and mitochondrial targeting sequence (MTS) indicated. D, immunoblot analysis of empty vector control and Strep-METTL8 purifications from transient transfections. The immunoblot was probed with anti-Twin-Strep and anti-FLAG antibodies. The input represents 1% of total. Percent (%) recovery represents the fraction of SARS2-FLAG recovered from the total input in the vector or METTL8 purification. E and F, immunoblot analysis of empty vector control, METTL8, and METTL6 purifications from transient transfections. The immunoblot was probed with anti-Twin-Strep and anti-SARS2 antibodies. Input represents 4% of total extract used for purification. Percent (%) recovery represents the fraction of SARS2 recovered from the total input in the vector, METTL8, and METTL6 purifications. *p < 0.05. p = 0.0232 for vector versus METTL8, 0.2089 for vector versus METTL6. METTL8, methyltransferase-like 8; PSM, peptide spectral matches.

Fig 5: METTL8 interacts with mt-tRNA-Ser-UGA and mt-tRNA-Thr-UGU.A, purification of METTL8 and analysis of protein and RNA. B, immunoblot analysis of Strep-Tactin purifications from human cells expressing control, METTL8, or METTL8-?MTS fused to the twin-Strep tag. The immunoblot was probed with anti-TwinStrep and anti-actin antibodies. C, nucleic acid stain of RNAs extracted from the indicated input or purified samples after denaturing PAGE. The migration pattern of 5.8S rRNA (~150 nt), 5S rRNA (~120 nt), and tRNAs (~70–80 nt) are denoted. D, Northern blot analysis of the gel in (C) using the indicated probes. Input represents 2% of total extracts used for purification. The percentage yield represents the amount of RNA in the Strep purification that was recovered from the total input. The experiment was repeated three times with comparable results. METTL8, methyltransferase-like 8; mt, mitochondrial.