tRNA methyltransferase (TRDMT1/DNMT2) participates in network of RNA modification in the Moss Physcomitrium patens.

Crosstalk among tRNA modification proteins have been implicated in important functional roles in biology. We previously reported that Physcomitrium tRNA (cytosine38-C5)-methyltransferase, TRDMT1/DNMT2 catalyses C38, C48 and C49 methylation in tRNAAsp(GUC) and plays a crucial role in regulating transcription/stability of tRNAAsp(GUC) under oxidative stress. To gain insight into its precise mode of regulation, in the present study we show that moss TRDMT1/DNMT2 participates in broader networks of tRNA modification pathways. Using in silico methods we first identified that the yeast homologs of m7G46 methyltransferase Trm8 that catalyses m7G46 methylation, its obligate partner protein Trm82 and the Queuine tRNA ribosyl transferase (QTRT1) exist in a functional network with PpTRDMT1/PpDNMT2. To examine genetic interaction between PpTRDMT1/PpDNMT2, Trm8-82 and QTRT1, PpTRDMT1/PpDNMT2 loss-of-function mutants (ppdnmt2) and ppdnmt2 rescued lines (PpDNMT2-c) were used and transcript abundance of each gene was examined by qRT-PCR in background of these plants. Thereafter, physical interaction between PpTrm8/8 L1/8 L2 and PpQTRT1 with specific catalytic motifs in PpTRDMT1/PpDNMT2 were studied by yeast two-hybrid assay. The observed protein-protein interaction was also supported by in silico analysis of docked complexes of stretches of PpTRDMT1/PpDNMT2 and the Trm8 homologs. Alanine scanning mutagenesis study identified Threonine 11 in PpTRDMT1/PpDNMT2 located close to catalytic motif IV to be crucial for stable complex formation between PpTRDMT1/PpDNMT2 and its binding partners. On the basis of the results obtained we propose that pathways leading to m7G46 modification by tRNA-Guanine-N-7-methyltransferases and incorporation of Queuine by QTRT1 may be modulated at transcriptional/post-transcriptional levels by PpTRDMT1/PpDNMT2 function in P. patens. SIGNIFICANCE STATEMENT: tRNA modifying enzymes form a network in the moss P. patens.