Mechanism of human tRNA 3’CCA maturation

Summary The non-templated addition of the 3’CCA end is the final universal step of tRNA maturation. In humans, 3’CCA addition on nuclear- (nu-tRNA) and mitochondria-encoded tRNAs (mt-tRNA) is catalyzed by a single CCA-adding enzyme, TRNT1, but its precise mechanism remains unknown. Here, we report structures of TRNT1 trapped at various stages during the 3’CCA addition cycle on canonical and non-canonical mt-tRNAs bound to the mitochondrial tRNA maturation platform TRMT10C-SDR5C1. Combined with biochemical data, these structures demonstrate that 3’CCA addition proceeds by a continuous polymerization and translocation mechanism, in which the growing RNA primer remodels the TRNT1 catalytic site to define the specificity of non-templated 3’CCA addition. Moreover, they reveal a relaxed recognition mode that allows TRNT1 to mature both canonical nu-tRNA and non-canonical mt-tRNA substrates. Finally, biochemical analyses of disease-associated TRNT1 variants provide insights into their molecular pathogenesis. Taken together, these results provide a detailed mechanistic picture of human tRNA-3’CCA maturation.