Fig. 2: METTL8 crosslinks to mt-tRNAs in cells.
a HEK293 cells expressing METTL8-His6-2xFLAG or the His6-2xFLAG tag were UV crosslinked. Protein-RNA complexes were retrieved, and co-purified RNAs trimmed, radioactively labelled and ligated to adaptors. Protein-RNA complexes were separated by denaturing PAGE, transferred to a nitrocellulose membrane and detected by autoradiography. Areas of the membrane excised are indicated with red boxes. Data presented in a–c and e derive from a single experiment. b RNAs eluted from the membrane areas indicated in (a) were reverse transcribed and the cDNA library deep sequenced. Doughnut charts show the relative distribution of reads derived from different classes of RNA in the His6-2xFLAG and METTL8-His6-2xFLAG samples. Abbreviations: mRNA - messenger RNA, tRNA - transfer RNA, lncRNA - long non-coding RNA, mt-tRNA - mitochondrial tRNA, mt-rRNA - mitochondrial rRNA, mt-mRNA - mitochondrial mRNA. c The normalised numbers of sequencing reads mapping to each mt-tRNA gene in the METTL8-His6-2xFLAG and His6-2xFLAG datasets are depicted as a heatmap in logarithmic scale (left panel). The fold-enrichment of reads derived from mt-tRNAs in the METTL8-His6-2xFLAG compared to His6-2xFLAG control is depicted in the right panel. d Lysates from crosslinked cells expressing METTL8-His6-2xFLAG or the His6-2×FLAG tag were used for immunoprecipitation experiments. Proteins and RNAs in input and eluate samples were analysed by western and northern blotting respectively. Three biologically independent experiments were performed and representative blots are shown. e The numbers of sequencing reads and nucleotide substitutions (Sub.) mapping to each nucleotide of the mt-RNAThr and mt-tRNASer(UCN) genes in the METTL8-His6-2xFLAG and control sample (His6-2xFLAG) are shown. The nucleotide sequence of each mt-tRNA is given with nucleotides of the anticodon stemloop (ASL) indicated in red. RPM - reads per million mapped reads. f, g Fluorescence anisotropy measurements were taken to determine the affinity of recombinant His14-MBP-METTL8 for different fluorescein-labelled oligonucleotides: ASLs of mt-tRNASer(UCN) and mt-tRNAMet (f), unstructured RNAs and DNA oligonucleotide (g). Data from three independent experiments are shown as mean ± standard deviation and dissociation constants (Kds) are given. Source data are provided as a Source Data file.
