Discrimination of initiator tRNA and start codon by mammalian mitochondrial initiation factor 3 in leaderless mRNA translation

The mammalian mitochondrial protein synthesis system produces thirteen essential subunits of oxidative phosphorylation (OXPHOS) complexes. Translation initiation in mammalian mitochondria is characterized by the use of leaderless mRNAs and non-AUG start codons, where the proofreading function of IF-3mt still remains elusive. Here, we developed a reconstituted mammalian mitochondrial translation system using in vitro transcribed and native mitochondrial tRNAs to investigate IF-3mt's proofreading function. Similar to bacterial IF-3, IF-3mt permits initiator tRNA to participate in initiation by discriminating the three G-C pairs in its anticodon stem, and the cognate interaction of its anticodon with the AUG start codon. As a result, IF-3mt promotes the accurate initiation of leaderless mRNAs. Nevertheless, IF-3mt can also facilitate initiation from the non-AUG(AUA) start codon through its unique N- and C-terminal extensions in concert with the 5-methylcytidine (m5C) or 5-formylcytidine (f5C) modification at the anticodon wobble position of mt-tRNAMet. This is partly because the IF-3mt-specific N- and C-terminal extensions and the KKGK-motif favor leaderless mRNA initiation and relax non-AUG start codon discrimination. Analyses of IF-3mt-depleted cells revealed that, in human cells, IF-3mt indeed participates in translating the ORFs of leaderless mRNAs, as well as the internal ORFs of bicistronic mRNAs. Overall design: Ribo-Seq and RNA-Seq were performed in Naïve and IF-3mt KO HEK293 cells.