Biochemical analysis of human tRNAHis guanylyltransferase in mitochondrial tRNAHis maturation

Mitochondria contain their own protein synthesis machinery, which includes mitochondrial tRNA maturation. It has been suggested that mammalian mitochondrial tRNAHis (mtRNAHis) is matured by post-transcriptional addition of guanosine at the −1 position (G−1), which serves as an identity element for mitochondrial histidyl-tRNA synthetase. However, the exact maturation process of mammalian mtRNAHis remains unclear. In cytoplasmic tRNAHis (ctRNAHis) maturation, tRNAHis guanylyltransferase (Thg1) adds a GTP onto the 5′-terminal of ctRNAHis and then removes the 5′-pyrophosphate to yield the mature 5′-monophospholylated G-1-ctRNAHis (pG-1-ctRNAHis). Although mammalian Thg1 is localized to both the cytoplasm and mitochondria, it remains unclear whether mammalian Thg1 plays a role in mtRNAHis maturation in mitochondria. Here, we demonstrated that human Thg1 (hThg1) catalyzes the G-1 addition reaction for both human ctRNAHis and mtRNAHis through recognition of the anticodon. While hThg1 catalyzed consecutive GTP additions to mtRNAHisin vitro, it did not exhibit any activity toward mature pG-1-mtRNAHis. We further found that hThg1 could add a GMP directly to the 5′-terminal of mtRNAHis in a template-dependent manner, but fungal Thg1 could not. Therefore, we hypothesized that acceleration of the pyrophosphate removal activity before or after the G-1 addition reaction is a key feature of hThg1 for maintaining a normal 5′-terminal of mtRNAHis in human mitochondria. This study provided a new insight into the differences between tRNAHis maturation in the cytoplasm and mitochondria of humans. •The mechanism of tRNAHis maturation in human mitochondria is proposed.•hThg1 catalyzes the addition of G-1 to both cytoplasmic and mitochondrial tRNAHis.•hThg1 incorporates multiple GTPs into immature mitochondrial tRNAHisin vitro.•hThg1 adds a GMP directly to mitochondrial tRNAHis in a template-dependent manner.