A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome

Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoribosome. Release-factor assays show that although ICT1 has retained its ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoribosomes

The invariant uridine of stop codons contacts the conserved NIKSR loop of human eRF1 in the ribosome

To unravel the region of human eukaryotic release factor 1 (eRF1) that is close to stop codons within the ribosome, we used mRNAs containing a single photoactivatable 4-thiouridine (s(4)U) residue in the first position of stop or control sense codons. Accurate phasing of these mRNAs onto the ribosome was achieved by the addition of tRNA(Asp). Under these conditions, eRF1 was shown to crosslink exclusively to mRNAs containing a stop or s(4)UGG codon. A procedure that yielded (32)P-labeled eRF1 deprived of the mRNA chain was developed; analysis of the labeled peptides generated after specific cleavage of both wild-type and mutant eRF1s maps the crosslink in the tripeptide KSR (positions 63–65 of human eRF1) and points to K63 located in the conserved NIKS loop as the main crosslinking site. These data directly show the interaction of the N-terminal (N) domain of eRF1 with stop codons within the 40S ribosomal subunit and provide strong support for the positioning of the eRF1 middle (M) domain on the 60S subunit. Thus, the N and M domains mimic the tRNA anticodon and acceptor arms, respectively

Optimal translational termination requires C4 lysyl hydroxylation of eRF1.

Efficient stop codon recognition and peptidyl-tRNA hydrolysis are essential in order to terminate translational elongation and maintain protein sequence fidelity. Eukaryotic translational termination is mediated by a release factor complex that includes eukaryotic release factor 1 (eRF1) and eRF3. The N terminus of eRF1 contains highly conserved sequence motifs that couple stop codon recognition at the ribosomal A site to peptidyl-tRNA hydrolysis. We reveal that Jumonji domain-containing 4 (Jmjd4), a 2-oxoglutarate- and Fe(II)-dependent oxygenase, catalyzes carbon 4 (C4) lysyl hydroxylation of eRF1. This posttranslational modification takes place at an invariant lysine within the eRF1 NIKS motif and is required for optimal translational termination efficiency. These findings further highlight the role of 2-oxoglutarate/Fe(II) oxygenases in fundamental cellular processes and provide additional evidence that ensuring fidelity of protein translation is a major role of hydroxylation