Poly(A)-binding protein (PABP) is a major component of the messenger RNA–protein complex. PABP is able to bind the poly(A) tail of mRNA, as well as translation initiation factor 4G and eukaryotic release factor 3a (eRF3a). PABP has been found to stimulate translation initiation and to inhibit nonsense-mediated mRNA decay. Using a reconstituted mammalian in vitro translation system, we show that PABP directly stimulates translation termination. PABP increases the efficiency of translation termination by recruitment of eRF3a and eRF1 to the ribosome. PABP's function in translation termination depends on its C-terminal domain and its interaction with the N-terminus of eRF3a. Interestingly, we discover that full-length eRF3a exerts a different mode of function compared to its truncated form eRF3c, which lacks the N-terminal domain. Pre-association of eRF3a, but not of eRF3c, with pre-termination complexes (preTCs) significantly increases the efficiency of peptidyl–tRNA hydrolysis by eRF1. This implicates new, additional interactions of full-length eRF3a with the ribosomal preTC. Based on our findings, we suggest that PABP enhances the productive binding of the eRF1–eRF3 complex to the ribosome, via interactions with the N-terminal domain of eRF3a which itself has an active role in translation termination.

SummaryEfficient 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.

Stabilization of the ribosomal complexes plays an important role in translational control. Mechanisms of ribosome stabilization have been studied in detail for initiation and elongation of eukaryotic translation, but almost nothing is known about stabilization of eukaryotic termination ribosomal complexes. Here, we present one of the mechanisms of fine-tuning of the translation termination process in eukaryotes. We show that certain deacylated tRNAs, remaining in the E site of the ribosome at the end of the elongation cycle, increase the stability of the termination and posttermination complexes. Moreover, only the part of eRF1 recognizing the stop codon is stabilized in the A site of the ribosome, and the stabilization is not dependent on the hydrolysis of peptidyl-tRNA. The determinants, defining this property of the tRNA, reside in the acceptor stem. It was demonstrated by site-directed mutagenesis of tRNAVal and construction of a mini-helix structure identical to the acceptor stem of tRNA. The mechanism of this stabilization is different from the fixation of the unrotated state of the ribosome by CCA end of tRNA or by cycloheximide in the E site. Our data allow to reveal the possible functions of the isodecoder tRNAs in eukaryotes.

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