Hetero Subunits Assembly Study of RNA Modification Enzyme by Wheat Germ Cell-Free Translation System

In the living cells, many kinds of modified nucleosides exist in various RNA species. These modified nucleosides are generated by specific RNA modification enzymes. In almost cases, RNA modification enzyme is composed by one single subunit or homo-subunits. However, recent studies have revealed that eukaryote tRNA (m⁷G46) methyltransferases are exceptionally constituted by hetero-subunits (Trm8/Trm82 in yeast; METTL1/WDR4 in human). This enzyme catalyzes the methyl-transfer from S-adenosyl-L-methionine to the N⁷ atom of the semi-conserved guanosine at position 46 in the extra-loop of tRNA. To clarify the functions of two subunits, we employed wheat germ cell-free translation system. When the Trm8 or Trm82 subunit alone was synthesized, methyl-transfer activity was not detectable. In contrast, when both Trm8 and Trm82 subunits were synthesized together, tRNA methyltransferase activity was clearly detected. Furthermore, we mixed two subunits after the synthesis, however formation of the active hetero-dimer was not observed. These results demonstrated that the formation of the active Trm8 and Trm82 hetero-dimer requires the subunit-subunit interaction during the protein synthesis