Application of mutational profiling: New functional analyses reveal the tRNA recognition mechanism of tRNA m(1)A22 methyltransferase

Transfer RNAs undergo diverse posttranscriptional modifications to regulate a myriad of cellular events including translation, stress response, and viral replication. These posttranscriptional modifications are synthesized by site-specific modification enzymes. Recent RNA-seq techniques have revealed multiple features of tRNA such as tRNA abundance, tRNA modification, and tRNA structure. Here, we adapt a tRNA-sequencing technique and design a new functional analysis where we perform mutational profiling of tRNA modifications to gain mechanistic insights into how tRNA modification enzymes recognize substrate tRNA. Profiling of Geobacillus stearothermophilus tRNAs and protein orthology analysis predict the existence of natural modifications in 44 tRNA molecular species of G. stearothermophilus. We selected the 1-methyladenosine modification at position 22 (m(1)A22) and tRNA (m(1)A22) methyltransferase (TrmK) for further analysis. Relative quantification of m(1)A22 levels in 59 tRNA transcripts by mutational profiling reveals that TrmK selectively methylates a subset of tRNAs. Using 240 variants of tRNA(Leu) transcripts, we demonstrate the conserved nucleosides including U8, A14, G15, G18, G19, U55, Purine57, and A58 are important for the methyl transfer reaction of TrmK. Additional biochemical experiments reveal that TrmK strictly recognizes U8, A14, G18, and U55 in tRNA. Furthermore, these findings from tRNA(Leu) variants were crossvalidated using variants of three different tRNA species. Finally, a model of the TrmK–tRNA complex structure was constructed based on our findings and previous biochemical and structural studies by others. Collectively, our study expands functional analyses of tRNA modification enzyme in a high-throughput manner where our assay rapidly identifies substrates from a large pool of tRNAs.