Mechanism of human tRNA 3'CCA maturation

Kuhle, B.; Krebs, L.; Bhatta, A.; Dennerlein, S.; Rehling, P.; Hillen, H.

doi:10.64898/2026.03.02.709036

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bioRxiv · 10.64898/2026.03.02.709036

Mechanism of human tRNA 3'CCA maturation

Kuhle, B.·Krebs, L.·Bhatta, A.·Dennerlein, S.·Rehling, P.·Hillen, H.

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Abstract

The non-templated addition of the 3CCA end is the final universal step of tRNA maturation. In humans, 3CCA 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 3CCA 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 3CCA 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 3CCA 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-3CCA maturation.

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Kuhle, B., Krebs, L., Bhatta, A., Dennerlein, S., Rehling, P., Hillen, H.. 2026-03-03. Mechanism of human tRNA 3'CCA maturation. https://doi.org/10.64898/2026.03.02.709036

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