PABP enhances release factor recruitment and stop codon recognition during translation termination

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

Structure of a human cap-dependent 48S translation pre-initiation complex

Eukaryotic translation initiation is tightly regulated, requiring a set of conserved initiation factors (eIFs). Translation of a capped mRNA depends on the trimeric eIF4F complex and eIF4B to load the mRNA onto the 43S pre-initiation complex comprising 40S and initiation factors 1, 1A, 2, 3 and 5 as well as initiator-tRNA. Binding of the mRNA is followed by mRNA scanning in the 48S pre-initiation complex, until a start codon is recognised. Here, we use a reconstituted system to prepare human 48S complexes assembled on capped mRNA in the presence of eIF4B and eIF4F. The highly purified h-48S complexes are used for cross-linking/mass spectrometry, revealing the protein interaction network in this complex. We report the electron cryo-microscopy structure of the h-48S complex at 6.3 Å resolution. While the majority of eIF4B and eIF4F appear to be flexible with respect to the ribosome, additional density is detected at the entrance of the 40S mRNA channel which we attribute to the RNA-recognition motif of eIF4B. The eight core subunits of eIF3 are bound at the 40S solvent-exposed side, as well as the subunits eIF3d, eIF3b and eIF3i. elF2 and initiator-tRNA bound to the start codon are present at the 40S intersubunit side. This cryo-EM structure represents a molecular snap-shot revealing the h-48S complex following start codon recognition

Caractérisation de complexes responsables de la dégradation des ARNm non-sens

Nonsense-mediated mRNA decay (NMD) is an important eukaryotic quality control mechanism that recognizes and degrades mRNA containing a premature termination codon (PTC). Up-frameshift proteins constitute the conserved core NMD factors (UPF1, UPF2 and UPF3). They mediate the recognition of a NMD substrate, i.e. a ribosome stalled at a PTC. UPF proteins were shown to associate with eukaryotic release factors (eRF1 and eRF3) and were suggested to impede translation termination. We showed that, at a normal termination codon, Poly(A)-binding protein (PABP) stimulates translation termination by directly interacting with eRF3a. Using a reconstituted in vitro translation system, we studied translation termination in the presence of the factors PABP and UPF1 using biochemistry and single particle electron cryo-microscopy (Cryo-EM). Additionally, we analysed the role of the other NMD factors UPF2 and UPF3B in translation termination in vitro. We discovered a novel role for UPF3B in translation termination. Moreover, we observed a novel interaction between UPF3B and the SMG1-8-9 kinase complex. The presence of UPF3B affects the kinase activity of SMG1 and thus the phosphorylation state of UPF1. Our results highlight a much more complex interplay of the NMD factors with the translation termination machinery and SMG1 kinase than anticipated.Le système de contrôle appelé dégradation des ARNm non-sens (NMD) permet de détecter puis de dégrader des ARNm contenant un codon de terminaison prématuré (PTC). Les facteurs principaux de la NMD : UPF1, UPF2 et UPF3 reconnaissent les PTCs en interagissant avec les facteurs de terminaison eRF1, eRF3 et la protéine Poly(A) binding (PABP). La reconstitution d’un système de traduction in vitro a permis d’étudier la terminaison de la traduction en présence des facteurs PABP et UPF1, à l’aide de méthodes de biochimie et de cryo-microscopie électronique. L’étude du rôle du facteur de NMD UPF3B dans la terminaison de la traduction a mis en évidence une double action de cette protéine ; tout d’abord, un retardement de la reconnaissance du codon stop et également la promotion de la dissociation du ribosome. Ce travail a également permis de mettre en évidence une nouvelle interaction entre UPF3B et la kinase SMG1-8-9 et de montrer comment cette interaction affecte l’état de phosphorylation de UPF1. Les résultats de cette étude montrent une interaction complexe entre les différents facteurs de NMD et la kinase SMG1

Characterization of Nonsense-mediated mRNA decay complexes

Le système de contrôle appelé dégradation des ARNm non-sens (NMD) permet de détecter puis de dégrader des ARNm contenant un codon de terminaison prématuré (PTC). Les facteurs principaux de la NMD : UPF1, UPF2 et UPF3 reconnaissent les PTCs en interagissant avec les facteurs de terminaison eRF1, eRF3 et la protéine Poly(A) binding (PABP). La reconstitution d’un système de traduction in vitro a permis d’étudier la terminaison de la traduction en présence des facteurs PABP et UPF1, à l’aide de méthodes de biochimie et de cryo-microscopie électronique. L’étude du rôle du facteur de NMD UPF3B dans la terminaison de la traduction a mis en évidence une double action de cette protéine ; tout d’abord, un retardement de la reconnaissance du codon stop et également la promotion de la dissociation du ribosome. Ce travail a également permis de mettre en évidence une nouvelle interaction entre UPF3B et la kinase SMG1-8-9 et de montrer comment cette interaction affecte l’état de phosphorylation de UPF1. Les résultats de cette étude montrent une interaction complexe entre les différents facteurs de NMD et la kinase SMG1.Nonsense-mediated mRNA decay (NMD) is an important eukaryotic quality control mechanism that recognizes and degrades mRNA containing a premature termination codon (PTC). Up-frameshift proteins constitute the conserved core NMD factors (UPF1, UPF2 and UPF3). They mediate the recognition of a NMD substrate, i.e. a ribosome stalled at a PTC. UPF proteins were shown to associate with eukaryotic release factors (eRF1 and eRF3) and were suggested to impede translation termination. We showed that, at a normal termination codon, Poly(A)-binding protein (PABP) stimulates translation termination by directly interacting with eRF3a. Using a reconstituted in vitro translation system, we studied translation termination in the presence of the factors PABP and UPF1 using biochemistry and single particle electron cryo-microscopy (Cryo-EM). Additionally, we analysed the role of the other NMD factors UPF2 and UPF3B in translation termination in vitro. We discovered a novel role for UPF3B in translation termination. Moreover, we observed a novel interaction between UPF3B and the SMG1-8-9 kinase complex. The presence of UPF3B affects the kinase activity of SMG1 and thus the phosphorylation state of UPF1. Our results highlight a much more complex interplay of the NMD factors with the translation termination machinery and SMG1 kinase than anticipated

Caractérisation de complexes responsables de la dégradation des ARNm non-sens

Nonsense-mediated mRNA decay (NMD) is an important eukaryotic quality control mechanism that recognizes and degrades mRNA containing a premature termination codon (PTC). Up-frameshift proteins constitute the conserved core NMD factors (UPF1, UPF2 and UPF3). They mediate the recognition of a NMD substrate, i.e. a ribosome stalled at a PTC. UPF proteins were shown to associate with eukaryotic release factors (eRF1 and eRF3) and were suggested to impede translation termination. We showed that, at a normal termination codon, Poly(A)-binding protein (PABP) stimulates translation termination by directly interacting with eRF3a. Using a reconstituted in vitro translation system, we studied translation termination in the presence of the factors PABP and UPF1 using biochemistry and single particle electron cryo-microscopy (Cryo-EM). Additionally, we analysed the role of the other NMD factors UPF2 and UPF3B in translation termination in vitro. We discovered a novel role for UPF3B in translation termination. Moreover, we observed a novel interaction between UPF3B and the SMG1-8-9 kinase complex. The presence of UPF3B affects the kinase activity of SMG1 and thus the phosphorylation state of UPF1. Our results highlight a much more complex interplay of the NMD factors with the translation termination machinery and SMG1 kinase than anticipated.Le système de contrôle appelé dégradation des ARNm non-sens (NMD) permet de détecter puis de dégrader des ARNm contenant un codon de terminaison prématuré (PTC). Les facteurs principaux de la NMD : UPF1, UPF2 et UPF3 reconnaissent les PTCs en interagissant avec les facteurs de terminaison eRF1, eRF3 et la protéine Poly(A) binding (PABP). La reconstitution d’un système de traduction in vitro a permis d’étudier la terminaison de la traduction en présence des facteurs PABP et UPF1, à l’aide de méthodes de biochimie et de cryo-microscopie électronique. L’étude du rôle du facteur de NMD UPF3B dans la terminaison de la traduction a mis en évidence une double action de cette protéine ; tout d’abord, un retardement de la reconnaissance du codon stop et également la promotion de la dissociation du ribosome. Ce travail a également permis de mettre en évidence une nouvelle interaction entre UPF3B et la kinase SMG1-8-9 et de montrer comment cette interaction affecte l’état de phosphorylation de UPF1. Les résultats de cette étude montrent une interaction complexe entre les différents facteurs de NMD et la kinase SMG1

Disability for Function: Loss of Ca²⁺-Binding Is Obligatory for Fitness of Mammalian βγ-Crystallins

Vertebrate βγ-crystallins belonging to the βγ-crystallin superfamily lack functional Ca²⁺-binding sites, while their microbial homologues do not; for example, three out of four sites in lens γ-crystallins are disabled. Such loss of Ca²⁺-binding function in non-lens βγ-crystallins from mammals (e.g., AIM1 and Crybg3) raises the possibility of a trade-off in the evolutionary extinction of Ca²⁺-binding. We test this hypothesis by reconstructing ancestral Ca²⁺-binding motifs (transforming disabled motifs into the canonical ones) in the lens γB-crystallin by introducing minimal sets of mutations. Upon incorporation of serine at the fifth position in the N/D-N/D-X-X-S/T(5)-S motif, which endowed a domain with microbial characteristics, a decreased domain stability was observed. Ca²⁺ further destabilized the N-terminal domain (NTD) and its serine mutants profoundly, while the incorporation of a C-terminal domain (CTD) nullified this destabilization. On the other hand, Ca²⁺-induced destabilization of the CTD was not rescued by the introduction of an NTD. Of note, only one out of four sites is functional in the NTD of γB-crystallins responsible for weak Ca²⁺ binding, but the deleterious effects of Ca²⁺ are overcome by introduction of a CTD. The rationale for the onset of cataracts by certain mutations, such as R77S, which have not been clarified by structural means, could be explained by this work. The findings presented here shed light on the evolutionary innovations in terms of the functional loss of Ca²⁺-binding and acquisition of a bilobed domain, besides imparting additional advantages (e.g., protection from light) required for specialized functions