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.

Toxic dipeptide-repeat (DPR) proteins are produced from expanded G4C2 repeats in the C9ORF72 gene, the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥20 repeats inhibit the ribosome’s peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryogenic electron microscopy (cryo-EM) reveals that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center (PTC). Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with poly-PR and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.

The human DEAD-box RNA-helicase DDX19 functions in mRNA export through the nuclear pore complex. The yeast homolog of this protein, Dbp5, has been reported to participate in translation termination. Using a reconstituted mammalian in vitro translation system, we show that the human protein DDX19 is also important for translation termination. It is associated with the fraction of translating ribosomes. We show that DDX19 interacts with pre-termination complexes (preTCs) in a nucleotide-dependent manner. Furthermore, DDX19 increases the efficiency of termination complex (TC) formation and the peptide release in the presence of eukaryotic release factors. Using the eRF1(AGQ) mutant protein or a non-hydrolysable analog of GTP to inhibit subsequent peptidyl-tRNA hydrolysis, we reveal that the activation of translation termination by DDX19 occurs during the stop codon recognition. This activation is a result of DDX19 binding to preTC and a concomitant stabilization of terminating ribosomes. Moreover, we show that DDX19 stabilizes ribosome complexes with translation elongation factors eEF1 and eEF2. Taken together, our findings reveal that the human RNA helicase DDX19 actively participates in protein biosynthesis.