Crystal-packing analysis of translation initiation factor 2 reveals new details of its function.

Eukaryotic and archaeal translation initiation factor 2 in complex with GTP delivers the initiator methionyl-tRNA to the small ribosomal subunit. Over the past 20 years, thanks to the efforts of various research groups, including ours, this factor from the archaeon Sulfolobus solfataricus and its individual subunits have been crystallized in ten different space groups. Analysis of the molecular packing in these crystals makes it possible to better understand the roles of functionally significant switches and other elements of the nucleotide-binding pocket during the function of the factor as well as the influence of external effects on its transition between active and inactive states.

Crystal structure of archaeal IF5A-DHS complex reveals insights into the hypusination mechanism.

The translation factor IF5A is highly conserved in Eukarya and Archaea and undergoes a unique post-translational hypusine modification by the deoxyhypusine synthase (DHS) enzyme. DHS transfers the butylamine moiety from spermidine to IF5A using NAD as a cofactor, forming a deoxyhypusine intermediate. IF5A is a key player in protein synthesis, preventing ribosome stalling in proline-rich sequences during translation elongation and facilitating translation elongation and termination. Additionally, human eIF5A participates in various essential cellular processes and contributes to cancer metastasis, with inhibiting hypusination showing anti-proliferative effects. The hypusination pathway of IF5A is therefore an attractive new therapeutic target. We elucidated the 2.0 Å X-ray crystal structure of the archaeal DHS-IF5A complex, revealing hetero-octameric architecture and providing a detailed view of the complex active site including the hypusination loop. This structure, along with biophysical data and molecular dynamics simulations, provides new insights into the catalytic mechanism of the hypusination reaction.

Structural insights into the evolution of late steps of translation initiation in the three domains of life.

In eukaryotes and in archaea late steps of translation initiation involve the two initiation factors e/aIF5B and e/aIF1A. These two factors are also orthologous to the bacterial IF2 and IF1 proteins, respectively. Recent cryo-EM studies showed how e/aIF5B and e/aIF1A cooperate on the small ribosomal subunit to favor the binding of the large ribosomal subunit and the formation of a ribosome competent for elongation. In this review, pioneering studies and recent biochemical and structural results providing new insights into the role of a/eIF5B in archaea and eukaryotes will be presented. Recent structures will also be compared to orthologous bacterial initiation complexes to highlight domain-specific features and the evolution of initiation mechanisms.

Backbone resonance assignments of the C-terminal region of human translation initiation factor eIF4B.

Translation initiation in eukaryotes is an early step in protein synthesis, requiring multiple factors to recruit the ribosomal small subunit to the mRNA 5' untranslated region. One such protein factor is the eukaryotic translation initiation factor 4B (eIF4B), which increases the activity of the eIF4A RNA helicase, and is linked to cell survival and proliferation. We report here the protein backbone chemical shift assignments corresponding to the C-terminal 279 residues of human eIF4B. Analysis of the chemical shift values identifies one main helical region in the area previously linked to RNA binding, and confirms that the overall C-terminal region is intrinsically disordered.

Cryo-EM structure of human eIF5A-DHS complex reveals the molecular basis of hypusination-associated neurodegenerative disorders.

Hypusination is a unique post-translational modification of the eukaryotic translation factor 5A (eIF5A) that is essential for overcoming ribosome stalling at polyproline sequence stretches. The initial step of hypusination, the formation of deoxyhypusine, is catalyzed by deoxyhypusine synthase (DHS), however, the molecular details of the DHS-mediated reaction remained elusive. Recently, patient-derived variants of DHS and eIF5A have been linked to rare neurodevelopmental disorders. Here, we present the cryo-EM structure of the human eIF5A-DHS complex at 2.8 Å resolution and a crystal structure of DHS trapped in the key reaction transition state. Furthermore, we show that disease-associated DHS variants influence the complex formation and hypusination efficiency. Hence, our work dissects the molecular details of the deoxyhypusine synthesis reaction and reveals how clinically-relevant mutations affect this crucial cellular process.

Cyst stem cell lineage eIF5 non-autonomously prevents testicular germ cell tumor formation via eIF1A/eIF2γ-mediated pre-initiation complex.

BACKGROUND: Stem cell niche maintains stem cell population identity and is essential for the homeostasis of self-renewal and differentiation in Drosophila testes. However, the mechanisms of CySC lineage signals-mediated soma-germline communications in response to external stimuli are unclear. METHODS: Pre-initiation complex functions were evaluated by UAS-Gal4-mediated cell effects. RNA sequencing was conducted in NC and eIF5 siRNA-treated cells. Genetic interaction analysis was used to indicate the relationships between eIF5 and eIF1A/eIF2γ in Drosophila testes. RESULTS: Here, we demonstrated that in CySCs, translation initiation factor eIF5 mediates cyst cell differentiation and the non-autonomously affected germ cell differentiation process. CySCs lacking eIF5 displayed unbalanced cell proliferation and apoptosis, forming testicular germ cell tumors (TGCTs) during spermatogenesis. eIF5 transcriptional regulation network analysis identified multiple metabolic processes and several key factors that might be involved in germ cell differentiation and TGCT formation. Importantly, knockdown of eIF1A and eIF2γ, key components of pre-initiation complex, mimicked the phenotype of knocking down eIF5 in the stem cell niche of Drosophila testes. Genetic interaction analysis indicated that eIF5 was sufficient to rescue the phenotype of tumorlike structures induced by down-regulating eIF1A or eIF2γ in CySCs. CONCLUSIONS: These findings demonstrated that CySC lineage eIF5, together with eIF1A or eIF2γ, mediates soma-germline communications for the stem cell niche homeostasis in Drosophila testes, providing new insights for the prevention of TGCTs.

Structure and Function of Archaeal Translation Initiation Factor 2 Fragments Containing Cys2-Cys2 Motifs.

The heterotrimeric (αßγ) translation initiation factor 2 of archaea and eukaryotes (a/eIF2) supplies the P-site of the ribosome with the initiation tRNA. Its two subunits (ß and γ) contain the Cys2-Cys2 motif, which is capable of forming a stable zinc finger structure in the presence of zinc ions. In this work, comparative analysis of the fragments containing Cys2-Cys2 motifs in the aIF2ß and aIF2γ structures from different organisms was carried out and their environments in crystals was analyzed. Based on the obtained data, a conclusion was made that the conformation and role of these fragments in the ß- and γ-subunits of the aIF2 are different.

Binding of guide piRNA triggers methylation of the unstructured N-terminal region of Aub leading to assembly of the piRNA amplification complex.

PIWI proteins use guide piRNAs to repress selfish genomic elements, protecting the genomic integrity of gametes and ensuring the fertility of animal species. Efficient transposon repression depends on amplification of piRNA guides in the ping-pong cycle, which in Drosophila entails tight cooperation between two PIWI proteins, Aub and Ago3. Here we show that post-translational modification, symmetric dimethylarginine (sDMA), of Aub is essential for piRNA biogenesis, transposon silencing and fertility. Methylation is triggered by loading of a piRNA guide into Aub, which exposes its unstructured N-terminal region to the PRMT5 methylosome complex. Thus, sDMA modification is a signal that Aub is loaded with piRNA guide. Amplification of piRNA in the ping-pong cycle requires assembly of a tertiary complex scaffolded by Krimper, which simultaneously binds the N-terminal regions of Aub and Ago3. To promote generation of new piRNA, Krimper uses its two Tudor domains to bind Aub and Ago3 in opposite modification and piRNA-loading states. Our results reveal that post-translational modifications in unstructured regions of PIWI proteins and their binding by Tudor domains that are capable of discriminating between modification states is essential for piRNA biogenesis and silencing.

The SARS-unique domain (SUD) of SARS-CoV and SARS-CoV-2 interacts with human Paip1 to enhance viral RNA translation.

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV-2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS-CoV-2 and SARS-CoV share an otherwise non-conserved part of non-structural protein 3 (Nsp3), therefore named as "SARS-unique domain" (SUD). We previously found a yeast-2-hybrid screen interaction of the SARS-CoV SUD with human poly(A)-binding protein (PABP)-interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS-CoV SUD:Paip1 interaction by size-exclusion chromatography, split-yellow fluorescent protein, and co-immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS-CoV-2 and Paip1. The three-dimensional structure of the N-terminal domain of SARS-CoV SUD ("macrodomain II", Mac2) in complex with the middle domain of Paip1, determined by X-ray crystallography and small-angle X-ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC-SARS-CoV replicon-transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS-CoV and SARS-CoV-2.

Blockade of EIF5A hypusination limits colorectal cancer growth by inhibiting MYC elongation.

Eukaryotic Translation Initiation Factor 5A (EIF5A) is a translation factor regulated by hypusination, a unique posttranslational modification catalyzed by deoxyhypusine synthetase (DHPS) and deoxyhypusine hydroxylase (DOHH) starting from the polyamine spermidine. Emerging data are showing that hypusinated EIF5A regulates key cellular processes such as autophagy, senescence, polyamine homeostasis, energy metabolism, and plays a role in cancer. However, the effects of EIF5A inhibition in preclinical cancer models, the mechanism of action, and specific translational targets are still poorly understood. We show here that hypusinated EIF5A promotes growth of colorectal cancer (CRC) cells by directly regulating MYC biosynthesis at specific pausing motifs. Inhibition of EIF5A hypusination with the DHPS inhibitor GC7 or through lentiviral-mediated knockdown of DHPS or EIF5A reduces the growth of various CRC cells. Multiplex gene expression analysis reveals that inhibition of hypusination impairs the expression of transcripts regulated by MYC, suggesting the involvement of this oncogene in the observed effect. Indeed, we demonstrate that EIF5A regulates MYC elongation without affecting its mRNA content or protein stability, by alleviating ribosome stalling at five distinct pausing motifs in MYC CDS. Of note, we show that blockade of the hypusination axis elicits a remarkable growth inhibitory effect in preclinical models of CRC and significantly reduces the size of polyps in APCMin/+ mice, a model of human familial adenomatous polyposis (FAP). Together, these data illustrate an unprecedented mechanism, whereby the tumor-promoting properties of hypusinated EIF5A are linked to its ability to regulate MYC elongation and provide a rationale for the use of DHPS/EIF5A inhibitors in CRC therapy.

Flexible NAD+ Binding in Deoxyhypusine Synthase Reflects the Dynamic Hypusine Modification of Translation Factor IF5A.

The eukaryotic and archaeal translation factor IF5A requires a post-translational hypusine modification, which is catalyzed by deoxyhypusine synthase (DHS) at a single lysine residue of IF5A with NAD+ and spermidine as cofactors, followed by hydroxylation to form hypusine. While human DHS catalyzed reactions have been well characterized, the mechanism of the hypusination of archaeal IF5A by DHS is not clear. Here we report a DHS structure from Pyrococcus horikoshii OT3 (PhoDHS) at 2.2 Å resolution. The structure reveals two states in a single functional unit (tetramer): two NAD+-bound monomers with the NAD+ and spermidine binding sites observed in multi-conformations (closed and open), and two NAD+-free monomers. The dynamic loop region V288-P299, in the vicinity of the active site, adopts different positions in the closed and open conformations and is disordered when NAD+ is absent. Combined with NAD+ binding analysis, it is clear that PhoDHS can exist in three states: apo, PhoDHS-2 equiv NAD+, and PhoDHS-4 equiv NAD+, which are affected by the NAD+ concentration. Our results demonstrate the dynamic structure of PhoDHS at the NAD+ and spermidine binding site, with conformational changes that may be the response to the local NAD+ concentration, and thus fine-tune the regulation of the translation process via the hypusine modification of IF5A.

Inhibition of Eukaryotic Translation Initiation Factor 5A (eIF5A) Hypusination Suppress p53 Translation and Alters the Association of eIF5A to the Ribosomes.

The eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for the viability of the cells whose proposed function is to prevent the stalling of the ribosomes during translation elongation. eIF5A activity requires a unique and functionally essential post-translational modification, the change of a lysine to hypusine. eIF5A is recognized as a promoter of cell proliferation, but it has also been suggested to induce apoptosis. To date, the precise molecular mechanism through which eIF5A affects these processes remains elusive. In the present study, we explored whether eIF5A is involved in controlling the stress-induced expression of the key cellular regulator p53. Our results show that treatment of HCT-116 colon cancer cells with the deoxyhypusine (DHS) inhibitor N1-guanyl-1,7-diamineheptane (GC7) caused both inhibition of eIF5A hypusination and a significant reduction of p53 expression in UV-treated cells, and that eIF5A controls p53 expression at the level of protein synthesis. Furthermore, we show that treatment with GC7 followed by UV-induced stress counteracts the pro-apoptotic process triggered by p53 up-regulation. More in general, the importance of eIF5A in the cellular stress response is illustrated by the finding that exposure to UV light promotes the binding of eIF5A to the ribosomes, whereas UV treatment complemented by the presence of GC7 inhibits such binding, allowing a decrease of de novo synthesis of p53 protein.

Crystal structure of the translation recovery factor Trf from Sulfolobus solfataricus.

During translation initiation, the heterotrimeric archaeal translation initiation factor 2 (aIF2) recruits the initiator tRNAi to the small ribosomal subunit. In the stationary growth phase and/or during nutrient stress, Sulfolobus solfataricus aIF2 has a second function: It protects leaderless mRNAs against degradation by binding to their 5'-ends. The S. solfataricus protein Sso2509 is a translation recovery factor (Trf) that interacts with aIF2 and is responsible for the release of aIF2 from bound mRNAs, thereby enabling translation re-initiation. It is a member of the domain of unknown function 35 (DUF35) protein family and is conserved in Sulfolobales as well as in other archaea. Here, we present the X-ray structure of S. solfataricus Trf solved to a resolution of 1.65 Å. Trf is composed of an N-terminal rubredoxin-like domain containing a bound zinc ion and a C-terminal oligosaccharide/oligonucleotide binding fold domain. The Trf structure reveals putative mRNA binding sites in both domains. Surprisingly, the Trf protein is structurally but not sequentially very similar to proteins linked to acyl-CoA utilization-for example, the Sso2064 protein from S. solfataricus-as well as to scaffold proteins found in the acetoacetyl-CoA thiolase/high-mobility group-CoA synthase complex of the archaeon Methanothermococcus thermolithotrophicus and in a steroid side-chain-cleaving aldolase complex from the bacterium Thermomonospora curvata. This suggests that members of the DUF35 protein family are able to act as scaffolding and binding proteins in a wide variety of biological processes.

The third structural switch in the archaeal translation initiation factor 2 (aIF2) molecule and its possible role in the initiation of GTP hydrolysis and the removal of aIF2 from the ribosome.

The structure of the γ subunit of archaeal translation initiation factor 2 (aIF2) from Sulfolobus solfataricus (SsoIF2γ) was determined in complex with GDPCP (a GTP analog). Crystals were obtained in the absence of magnesium ions in the crystallization solution. They belonged to space group P1, with five molecules in the unit cell. Four of these molecules are related in pairs by a common noncrystallographic twofold symmetry axis, while the fifth has no symmetry equivalent. Analysis of the structure and its comparison with other known aIF2 γ-subunit structures in the GTP-bound state show that (i) the magnesium ion is necessary for the formation and the maintenance of the active form of SsoIF2γ and (ii) in addition to the two previously known structural switches 1 and 2, eukaryotic translation initiation factor 2 (eIF2) and aIF2 molecules have another flexible region (switch 3), the function of which may consist of initiation of the hydrolysis of GTP and the removal of e/aIF2 from the ribosome after codon-anticodon recognition.

Polyadenylate-binding protein-interacting proteins PAIP1 and PAIP2 affect translation termination.

Polyadenylate-binding protein (PABP) stimulates translation termination via interaction of its C-terminal domain with eukaryotic polypeptide chain release factor, eRF3. Additionally, two other proteins, poly(A)-binding protein-interacting proteins 1 and 2 (PAIP1 and PAIP2), bind the same domain of PABP and regulate its translation-related activity. To study the biochemistry of eRF3 and PAIP1/2 competition for PABP binding, we quantified the effects of PAIPs on translation termination in the presence or absence of PABP. Our results demonstrated that both PAIP1 and PAIP2 prevented translation termination at the premature termination codon, by controlling PABP activity. Moreover, PAIP1 and PAIP2 inhibited the activity of free PABP on translation termination in vitro However, after binding the poly(A) tail, PABP became insensitive to suppression by PAIPs and efficiently activated translation termination in the presence of eRF3a. Additionally, we revealed that PAIP1 binds eRF3 in solution, which stabilizes the post-termination complex. These results indicated that PAIP1 and PAIP2 participate in translation termination and are important regulators of readthrough at the premature termination codon.

Structural basis for the nuclear import and export functions of the biportin Pdr6/Kap122.

Importins ferry proteins into nuclei while exportins carry cargoes to the cytoplasm. In the accompanying paper in this issue (Vera Rodriguez et al. 2019. J. Cell Biol. https://doi.org/10.1083/jcb.201812091), we discovered that Pdr6 is a biportin that imports, e.g., the SUMO E2 ligase Ubc9 while depleting the translation factor eIF5A from the nuclear compartment. In this paper, we report the structures of key transport intermediates, namely, of the Ubc9•Pdr6 import complex, of the RanGTP•Pdr6 heterodimer, and of the trimeric RanGTP•Pdr6•eIF5A export complex. These revealed nonlinear transport signals, chaperone-like interactions, and how the RanGTPase system drives Pdr6 to transport Ubc9 and eIF5A in opposite directions. The structures also provide unexpected insights into the evolution of transport selectivity. Specifically, they show that recognition of Ubc9 by Pdr6 differs fundamentally from that of the human Ubc9-importer Importin 13. Likewise, Pdr6 recognizes eIF5A in a nonhomologous manner compared with the mammalian eIF5A-exporter Exportin 4. This suggests that the import of Ubc9 and active nuclear exclusion of eIF5A evolved in different eukaryotic lineages more than once and independently from each other.

Leishmania infantum LeIF and its recombinant polypeptides induce the maturation of dendritic cells in vitro: An insight for dendritic cells based vaccine.

We previously showed that recombinant Leishmania infantum eukaryotic initiation factor (LieIF) was able to induce the secretion of cytokines IL-12, IL-10 and TNF-α by human monocytes. In this study, we explored in vitro the potential of LieIF to induce phenotypic maturation and functional differentiation of murine bone-marrow derived dendritic cells (BM-DCs). Moreover, in order to identify potential immunnomodulatory regions of LieIF, eight recombinant overlapping protein fragments covering the whole amino acid sequence of protein, were constructed and assessed in vitro for their ability to induce maturation of BM-DCs. Our data showed that LieIF and some of its recombinant polypeptides were able to induce elevated expression of CD40, CD80 and CD86 co-stimulatory molecules with concurrent IL-12 production. Moreover, we used an in vivo experimental model of cutaneous leishmaniasis consisted of susceptible Leishmania major-infected BALB/c mice and we demonstrated that LieIF-pulsed-BM-DCs adoptively transferred in mice were capable to confer protection against a high dose parasite challenge. This study further describes the immunomodulatory properties of LieIF and its polypeptides bringing relevant information for their exploitation as candidate molecules for vaccine development against leishmaniasis.

Ribosomal mutation in helix 32 of 18S rRNA alters fidelity of eukaryotic translation start site selection.

The 40S ribosome plays a critical role in start codon selection. To gain insights into the role of its 18S rRNA in start codon selection, a suppressor screen was performed that suppressed the preferential UUG start codon recognition (Suppressor of initiation codon: Sui- phenotype) associated with the eIF5G31R mutant. The C1209U mutation in helix h32 of 18S rRNA was found to suppress the Sui- and Gcn- (failure to derepress GCN4 expression) phenotype of the eIF5G31R mutant. The C1209U mutation suppressed Sui- and Gcd- (constitutive derepression of GCN4 expression) phenotype of eIF2ßS264Y , eIF1K60E , and eIF1A-ΔC mutation. We propose that the C1209U mutation in 40S ribosomal may perturb the premature head rotation in 'Closed/PIN ' state and enhance the stringency of translation start site selection.

Start Codon Recognition in Eukaryotic and Archaeal Translation Initiation: A Common Structural Core.

Understanding molecular mechanisms of ribosomal translation sheds light on the emergence and evolution of protein synthesis in the three domains of life. Universally, ribosomal translation is described in three steps: initiation, elongation and termination. During initiation, a macromolecular complex assembled around the small ribosomal subunit selects the start codon on the mRNA and defines the open reading frame. In this review, we focus on the comparison of start codon selection mechanisms in eukaryotes and archaea. Eukaryotic translation initiation is a very complicated process, involving many initiation factors. The most widespread mechanism for the discovery of the start codon is the scanning of the mRNA by a pre-initiation complex until the first AUG codon in a correct context is found. In archaea, long-range scanning does not occur because of the presence of Shine-Dalgarno (SD) sequences or of short 5' untranslated regions. However, archaeal and eukaryotic translation initiations have three initiation factors in common: e/aIF1, e/aIF1A and e/aIF2 are directly involved in the selection of the start codon. Therefore, the idea that these archaeal and eukaryotic factors fulfill similar functions within a common structural ribosomal core complex has emerged. A divergence between eukaryotic and archaeal factors allowed for the adaptation to the long-range scanning process versus the SD mediated prepositioning of the ribosome.

Recessive Rare Variants in Deoxyhypusine Synthase, an Enzyme Involved in the Synthesis of Hypusine, Are Associated with a Neurodevelopmental Disorder.

Hypusine is formed post-translationally from lysine and is found in a single cellular protein, eukaryotic translation initiation factor-5A (eIF5A), and its homolog eIF5A2. Biosynthesis of hypusine is a two-step reaction involving the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). eIF5A is highly conserved throughout eukaryotic evolution and plays a role in mRNA translation, cellular proliferation, cellular differentiation, and inflammation. DHPS is also highly conserved and is essential for life, as Dhps-null mice are embryonic lethal. Using exome sequencing, we identified rare biallelic, recurrent, predicted likely pathogenic variants in DHPS segregating with disease in five affected individuals from four unrelated families. These individuals have similar neurodevelopmental features that include global developmental delay and seizures. Two of four affected females have short stature. All five affected individuals share a recurrent missense variant (c.518A>G [p.Asn173Ser]) in trans with a likely gene disrupting variant (c.1014+1G>A, c.912_917delTTACAT [p.Tyr305_Ile306del], or c.1A>G [p.Met1?]). cDNA studies demonstrated that the c.1014+1G>A variant causes aberrant splicing. Recombinant DHPS enzyme harboring either the p.Asn173Ser or p.Tyr305_Ile306del variant showed reduced (20%) or absent in vitro activity, respectively. We co-transfected constructs overexpressing HA-tagged DHPS (wild-type or mutant) and GFP-tagged eIF5A into HEK293T cells to determine the effect of these variants on hypusine biosynthesis and observed that the p.Tyr305_Ile306del and p.Asn173Ser variants resulted in reduced hypusination of eIF5A compared to wild-type DHPS enzyme. Our data suggest that rare biallelic variants in DHPS result in reduced enzyme activity that limits the hypusination of eIF5A and are associated with a neurodevelopmental disorder.