Boric acid intercepts 80S ribosome migration from AUG-stop by stabilizing eRF1.

In response to environmental changes, cells flexibly and rapidly alter gene expression through translational controls. In plants, the translation of NIP5;1, a boric acid diffusion facilitator, is downregulated in response to an excess amount of boric acid in the environment through upstream open reading frames (uORFs) that consist of only AUG and stop codons. However, the molecular details of how this minimum uORF controls translation of the downstream main ORF in a boric acid-dependent manner have remained unclear. Here, by combining ribosome profiling, translation complex profile sequencing, structural analysis with cryo-electron microscopy and biochemical assays, we show that the 80S ribosome assembled at AUG-stop migrates into the subsequent RNA segment, followed by downstream translation initiation, and that boric acid impedes this process by the stable confinement of eukaryotic release factor 1 on the 80S ribosome on AUG-stop. Our results provide molecular insight into translation regulation by a minimum and environment-responsive uORF.

Protection of eIF2B from inhibitory phosphorylated eIF2: A viral strategy to maintain mRNA translation during the PKR-triggered integrated stress response.

The integrated stress response (ISR) protects cells from a variety of insults. Once elicited (e.g., by virus infections), it eventually leads to the block of mRNA translation. Central to the ISR are the interactions between translation initiation factors eIF2 and eIF2B. Under normal conditions, eIF2 drives the initiation of protein synthesis through hydrolysis of GTP, which becomes replenished by the guanine nucleotide exchange factor eIF2B. The antiviral branch of the ISR is activated by the RNA-activated kinase PKR which phosphorylates eIF2, thereby converting it into an eIF2B inhibitor. Here, we describe the recently solved structures of eIF2B in complex with eIF2 and a novel escape strategy used by viruses. While unphosphorylated eIF2 interacts with eIF2B in its "productive" conformation, phosphorylated eIF2 [eIF2(αP)] engages a different binding cavity on eIF2B and forces it into the "nonproductive" conformation that prohibits guanine nucleotide exchange factor activity. It is well established that viruses express so-called PKR antagonists that interfere with double-strand RNA, PKR itself, or eIF2. However recently, three taxonomically unrelated viruses were reported to encode antagonists targeting eIF2B instead. For one antagonist, the S segment nonstructural protein of Sandfly fever Sicilian virus, atomic structures showed that it occupies the eIF2(αP)-binding cavity on eIF2B without imposing a switch to the nonproductive conformation. S segment nonstructural protein thus antagonizes the activity of PKR by protecting eIF2B from inhibition by eIF2(αP). As the ISR and specifically eIF2B are central to neuroprotection and a wide range of genetic and age-related diseases, these developments may open new possibilities for treatments.

Nondomain biopolymers: Flexible molecular strategies to acquire biological functions.

A long-standing assumption in molecular biology posits that the conservation of protein and nucleic acid sequences emphasizes the functional significance of biomolecules. These conserved sequences fold into distinct secondary and tertiary structures, enable highly specific molecular interactions, and regulate complex yet organized molecular processes within living cells. However, recent evidence suggests that biomolecules can also function through primary sequence regions that lack conservation across species or gene families. These regions typically do not form rigid structures, and their inherent flexibility is critical for their functional roles. This review examines the emerging roles and molecular mechanisms of "nondomain biomolecules," whose functions are not easily predicted due to the absence of conserved functional domains. We propose the hypothesis that both domain- and nondomain-type molecules work together to enable flexible and efficient molecular processes within the highly crowded intracellular environment.

A parasitic fungus employs mutated eIF4A to survive on rocaglate-synthesizing Aglaia plants.

Plants often generate secondary metabolites as defense mechanisms against parasites. Although some fungi may potentially overcome the barrier presented by antimicrobial compounds, only a limited number of examples and molecular mechanisms of resistance have been reported. Here, we found an Aglaia plant-parasitizing fungus that overcomes the toxicity of rocaglates, which are translation inhibitors synthesized by the plant, through an amino acid substitution in a eukaryotic translation initiation factor (eIF). De novo transcriptome assembly revealed that the fungus belongs to the Ophiocordyceps genus and that its eIF4A, a molecular target of rocaglates, harbors an amino acid substitution critical for rocaglate binding. Ribosome profiling harnessing a cucumber-infecting fungus, Colletotrichum orbiculare, demonstrated that the translational inhibitory effects of rocaglates were largely attenuated by the mutation found in the Aglaia parasite. The engineered C. orbiculare showed a survival advantage on cucumber plants with rocaglates. Our study exemplifies a plant-fungus tug-of-war centered on secondary metabolites produced by host plants.

Although plants may seem like passive creatures, they are in fact engaged in a constant battle against the parasitic fungi that attack them. To combat these fungal foes, plants produce small molecules that act like chemical weapons and kill the parasite. However, the fungi sometimes fight back, often by developing enzymes that can break down the deadly chemicals into harmless products. One class of anti-fungal molecules that has drawn great interest is rocaglates, as they show promise as treatments for cancer and COVID-19. Rocaglates are produced by plants in the Aglaia family and work by targeting the fungal molecule eIF4A which is fundamental for synthesizing proteins. Since proteins perform most of the chemistry necessary for life, one might think that rocaglates could ward off any fungus. But Chen et al. discovered there is in fact a species of fungi that can evade this powerful defense mechanism. After seeing this new-found fungal species successfully growing on Aglaia plants, Chen et al. set out to find how it is able to protect itself from rocoglates. Genetic analysis of the fungus revealed that its eIF4A contained a single mutation that 'blocked' rocaglates from interacting with it. Chen et al. confirmed this effect by engineering a second fungal species (which infects cucumber plants) so that its elF4A protein contained the mutation found in the new fungus. Fungi with the mutated eIF4A thrived on cucumber leaves treated with a chemical derived from rocaglates, whereas fungi with the non-mutated version were less successful. These results shed new light on the constant 'arms race' between plants and their fungal parasites, with each side evolving more sophisticated ways to overcome the other's defenses. Chen et al. hope that identifying the new rocaglate-resistant eIF4A mutation will help guide the development and use of any therapies based on rocaglates. Further work investigating how often the mutation occurs in humans will also be important for determining how effective these therapies will be.

METTL18-mediated histidine methylation of RPL3 modulates translation elongation for proteostasis maintenance.

Protein methylation occurs predominantly on lysine and arginine residues, but histidine also serves as a methylation substrate. However, a limited number of enzymes responsible for this modification have been reported. Moreover, the biological role of histidine methylation has remained poorly understood to date. Here, we report that human METTL18 is a histidine methyltransferase for the ribosomal protein RPL3 and that the modification specifically slows ribosome traversal on Tyr codons, allowing the proper folding of synthesized proteins. By performing an in vitro methylation assay with a methyl donor analog and quantitative mass spectrometry, we found that His245 of RPL3 is methylated at the τ-N position by METTL18. Structural comparison of the modified and unmodified ribosomes showed stoichiometric modification and suggested a role in translation reactions. Indeed, genome-wide ribosome profiling and an in vitro translation assay revealed that translation elongation at Tyr codons was suppressed by RPL3 methylation. Because the slower elongation provides enough time for nascent protein folding, RPL3 methylation protects cells from the cellular aggregation of Tyr-rich proteins. Our results reveal histidine methylation as an example of a ribosome modification that ensures proteome integrity in cells.

Conformational alterations in unidirectional ion transport of a light-driven chloride pump revealed using X-ray free electron lasers.

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.

The landscape of translational stall sites in bacteria revealed by monosome and disome profiling.

Ribosome pauses are associated with various cotranslational events and determine the fate of mRNAs and proteins. Thus, the identification of precise pause sites across the transcriptome is desirable; however, the landscape of ribosome pauses in bacteria remains ambiguous. Here, we harness monosome and disome (or collided ribosome) profiling strategies to survey ribosome pause sites in Escherichia coli Compared to eukaryotes, ribosome collisions in bacteria showed remarkable differences: a low frequency of disomes at stop codons, collisions occurring immediately after 70S assembly on start codons, and shorter queues of ribosomes trailing upstream. The pause sites corresponded with the biochemical validation by integrated nascent chain profiling (iNP) to detect polypeptidyl-tRNA, an elongation intermediate. Moreover, the subset of those sites showed puromycin resistance, presenting slow peptidyl transfer. Among the identified sites, the ribosome pause at Asn586 of ycbZ was validated by biochemical reporter assay, tRNA sequencing (tRNA-seq), and cryo-electron microscopy (cryo-EM) experiments. Our results provide a useful resource for ribosome stalling sites in bacteria.

eIF2B-capturing viral protein NSs suppresses the integrated stress response.

Various stressors such as viral infection lead to the suppression of cap-dependent translation and the activation of the integrated stress response (ISR), since the stress-induced phosphorylated eukaryotic translation initiation factor 2 [eIF2(αP)] tightly binds to eIF2B to prevent it from exchanging guanine nucleotide molecules on its substrate, unphosphorylated eIF2. Sandfly fever Sicilian virus (SFSV) evades this cap-dependent translation suppression through the interaction between its nonstructural protein NSs and host eIF2B. However, its precise mechanism has remained unclear. Here, our cryo-electron microscopy (cryo-EM) analysis reveals that SFSV NSs binds to the α-subunit of eIF2B in a competitive manner with eIF2(αP). Together with SFSV NSs, eIF2B retains nucleotide exchange activity even in the presence of eIF2(αP), in line with the cryo-EM structures of the eIF2B•SFSV NSs•unphosphorylated eIF2 complex. A genome-wide ribosome profiling analysis clarified that SFSV NSs expressed in cultured human cells attenuates the ISR triggered by thapsigargin, an endoplasmic reticulum stress inducer. Furthermore, SFSV NSs introduced in rat hippocampal neurons and human induced-pluripotent stem (iPS) cell-derived motor neurons exhibits neuroprotective effects against the ISR-inducing stress. Since ISR inhibition is beneficial in various neurological disease models, SFSV NSs may be a promising therapeutic ISR inhibitor.

Incorporation of Halogenated Amino Acids into Antibody Fragments at Multiple Specific Sites Enhances Antigen Binding.

Expansion of the amino-acid repertoire with synthetic derivatives introduces novel structures and functionalities into proteins. In this study, we improved the antigen binding of antibodies by incorporating halogenated tyrosines at multiple selective sites. Tyrosines in the Fab fragment of an anti-EGF-receptor antibody 059-152 were systematically replaced with 3-bromo- and 3-chlorotyrosines, and simultaneous replacements at four specific sites were found to cause a tenfold increase in the affinity toward the antigen. Structure modeling suggested that this effect was due to enhanced shape complementarity between the antigen and antibody molecules. On the other hand, we showed that chlorination in the constant domain, far from the binding interface, of Rituximab Fab also increased the affinity significantly (up to 17-fold). Our results showed that antigen binding is tunable with the halogenation in and out of the binding motifs.

Dual targeting of DDX3 and eIF4A by the translation inhibitor rocaglamide A.

The translation inhibitor rocaglamide A (RocA) has shown promising antitumor activity because it uniquely clamps eukaryotic initiation factor (eIF) 4A onto polypurine RNA for selective translational repression. As eIF4A has been speculated to be a unique target of RocA, alternative targets have not been investigated. Here, we reveal that DDX3 is another molecular target of RocA. Proximity-specific fluorescence labeling of an O-nitrobenzoxadiazole-conjugated derivative revealed that RocA binds to DDX3. RocA clamps the DDX3 protein onto polypurine RNA in an ATP-independent manner. Analysis of a de novo-assembled transcriptome from the plant Aglaia, a natural source of RocA, uncovered the amino acid critical for RocA binding. Moreover, ribosome profiling showed that because of the dominant-negative effect of RocA, high expression of eIF4A and DDX3 strengthens translational repression in cancer cells. This study indicates that sequence-selective clamping of DDX3 and eIF4A, and subsequent dominant-negative translational repression by RocA determine its tumor toxicity.

ISRIB Blunts the Integrated Stress Response by Allosterically Antagonising the Inhibitory Effect of Phosphorylated eIF2 on eIF2B.

The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B's nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB's efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(αP) and ISRIB reciprocally opposed each other's binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.

eIF2B and the Integrated Stress Response: A Structural and Mechanistic View.

The eukaryotic translation initiation factor eIF2 is a GTPase, which brings the initiator Met-tRNAi to the ribosome as the eIF2-GTP·Met-tRNAi ternary complex (TC). TC regeneration is catalyzed by the guanine nucleotide exchange factor (GEF) eIF2B. eIF2 phosphorylation by several stress-induced kinases converts it into a competitive inhibitor of eIF2B. Inhibition of eIF2B activity lowers cellular TC concentrations, which in turn triggers the integrated stress response (ISR). Depending on its degree of activation and duration, the ISR protects the cell from the stress or can itself induce apoptosis. ISR dysregulation is a causative factor in the pathology of multiple neurodegenerative disorders, while ISR inhibitors are neuroprotective. The realization that eIF2B is a promising therapeutic target has triggered significant interest in its structure and its mechanisms of action and regulation. Recently, four groups published the cryo-electron microscopy structures of eIF2B with its substrate eIF2 and/or its inhibitor, phosphorylated eIF2 [eIF2(α-P)]. While all three structures of the nonproductive eIF2B·eIF2(α-P) complex are similar to each other, there is a sharp disagreement between the published structures of the productive eIF2B·eIF2 complex. One group reports a structure similar to that of the nonproductive complex, whereas two others observe a vastly different eIF2B·eIF2 complex. Here, we discuss the recent reports on the structure, function, and regulation of eIF2B; the preclinical data on the use of ISR inhibitors for the treatment of neurodegenerative disorders; and how the new structural and biochemical information can inform and influence the use of eIF2B as a therapeutic target.

Glial pathology in a novel spontaneous mutant mouse of the Eif2b5 gene: a vanishing white matter disease model.

Vanishing white matter disease (VWM) is an autosomal recessive neurological disorder caused by mutation(s) in any subunit of eukaryotic translation initiation factor 2B (eIF2B), an activator of translation initiation factor eIF2. VWM occurs with mutation of the genes encoding eIF2B subunits (EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5). However, little is known regarding the underlying pathogenetic mechanisms or how to treat patients with VWM. Here we describe the identification and detailed analysis of a new spontaneous mutant mouse harboring a point mutation in the Eif2b5 gene (p.Ile98Met). Homozygous Eif2b5I98M mutant mice exhibited a small body, abnormal gait, male and female infertility, epileptic seizures, and a shortened lifespan. Biochemical analyses indicated that the mutant eIF2B protein with the Eif2b5I98M mutation decreased guanine nucleotide exchange activity on eIF2, and the level of the endoplasmic reticulum stress marker activating transcription factor 4 was elevated in the 1-month-old Eif2b5I98M brain. Histological analyses indicated up-regulated glial fibrillary acidic protein immunoreactivity in the astrocytes of the Eif2b5I98M forebrain and translocation of Bergmann glia in the Eif2b5I98M cerebellum, as well as increased mRNA expression of an endoplasmic reticulum stress marker, C/EBP homologous protein. Disruption of myelin and clustering of oligodendrocyte progenitor cells were also indicated in the white matter of the Eif2b5I98M spinal cord at 8 months old. Our data show that Eif2b5I98M mutants are a good model for understanding VWM pathogenesis and therapy development. Cover Image for this issue: doi: 10.1111/jnc.14751.

Structural basis for eIF2B inhibition in integrated stress response.

A core event in the integrated stress response, an adaptive pathway common to all eukaryotic cells in response to various stress stimuli, is the phosphorylation of eukaryotic translation initiation factor 2 (eIF2). Normally, unphosphorylated eIF2 transfers the methionylated initiator tRNA to the ribosome in a guanosine 5'-triphosphate-dependent manner. By contrast, phosphorylated eIF2 inhibits its specific guanine nucleotide exchange factor, eIF2B. To elucidate how the eIF2 phosphorylation status regulates the eIF2B activity, we determined cryo-electron microscopic and crystallographic structures of eIF2B in complex with unphosphorylated or phosphorylated eIF2. The unphosphorylated and phosphorylated forms of eIF2 bind to eIF2B in completely different manners: the nucleotide exchange-active and -inactive modes, respectively. These structures explain how phosphorylated eIF2 dominantly inhibits the nucleotide exchange activity of eIF2B.

HCV IRES Captures an Actively Translating 80S Ribosome.

Translation initiation of hepatitis C virus (HCV) genomic RNA is induced by an internal ribosome entry site (IRES). Our cryoelectron microscopy (cryo-EM) analysis revealed that the HCV IRES binds to the solvent side of the 40S platform of the cap-dependently translating 80S ribosome. Furthermore, we obtained the cryo-EM structures of the HCV IRES capturing the 40S subunit of the IRES-dependently translating 80S ribosome. In the elucidated structures, the HCV IRES "body," consisting of domain III except for subdomain IIIb, binds to the 40S subunit, while the "long arm," consisting of domain II, remains flexible and does not impede the ongoing translation. Biochemical experiments revealed that the cap-dependently translating ribosome becomes a better substrate for the HCV IRES than the free ribosome. Therefore, the HCV IRES is likely to efficiently induce the translation initiation of its downstream mRNA with the captured translating ribosome as soon as the ongoing translation terminates.

The Translation Inhibitor Rocaglamide Targets a Bimolecular Cavity between eIF4A and Polypurine RNA.

A class of translation inhibitors, exemplified by the natural product rocaglamide A (RocA), isolated from Aglaia genus plants, exhibits antitumor activity by clamping eukaryotic translation initiation factor 4A (eIF4A) onto polypurine sequences in mRNAs. This unusual inhibitory mechanism raises the question of how the drug imposes sequence selectivity onto a general translation factor. Here, we determined the crystal structure of the human eIF4A1⋅ATP analog⋅RocA⋅polypurine RNA complex. RocA targets the "bi-molecular cavity" formed characteristically by eIF4A1 and a sharply bent pair of consecutive purines in the RNA. Natural amino acid substitutions found in Aglaia eIF4As changed the cavity shape, leading to RocA resistance. This study provides an example of an RNA-sequence-selective interfacial inhibitor fitting into the space shaped cooperatively by protein and RNA with specific sequences.

Dynamic interaction of poly(A)-binding protein with the ribosome.

Eukaryotic mRNA has a cap structure and a poly(A) tail at the 5' and 3' ends, respectively. The cap structure is recognized by eIF (eukaryotic translation initiation factor) 4 F, while the poly(A) tail is bound by poly(A)-binding protein (PABP). PABP has four RNA recognition motifs (RRM1-4), and RRM1-2 binds both the poly(A) tail and eIF4G component of eIF4F, resulting in enhancement of translation. Here, we show that PABP interacts with the 40S and 60S ribosomal subunits dynamically via RRM2-3 or RRM3-4. Using a reconstituted protein expression system, we demonstrate that wild-type PABP activates translation in a dose-dependent manner, while a PABP mutant that binds poly(A) RNA and eIF4G, but not the ribosome, fails to do so. From these results, functional significance of the interaction of PABP with the ribosome is discussed.

JQ1 affects BRD2-dependent and independent transcription regulation without disrupting H4-hyperacetylated chromatin states.

The bromodomain and extra-terminal domain (BET) proteins are promising drug targets for cancer and immune diseases. However, BET inhibition effects have been studied more in the context of bromodomain-containing protein 4 (BRD4) than BRD2, and the BET protein association to histone H4-hyperacetylated chromatin is not understood at the genome-wide level. Here, we report transcription start site (TSS)-resolution integrative analyses of ChIP-seq and transcriptome profiles in human non-small cell lung cancer (NSCLC) cell line H23. We show that di-acetylation at K5 and K8 of histone H4 (H4K5acK8ac) co-localizes with H3K27ac and BRD2 in the majority of active enhancers and promoters, where BRD2 has a stronger association with H4K5acK8ac than H3K27ac. Although BET inhibition by JQ1 led to complete reduction of BRD2 binding to chromatin, only local changes of H4K5acK8ac levels were observed, suggesting that recruitment of BRD2 does not influence global histone H4 hyperacetylation levels. This finding supports a model in which recruitment of BET proteins via histone H4 hyperacetylation is predominant over hyperacetylation of histone H4 by BET protein-associated acetyltransferases. In addition, we found that a remarkable number of BRD2-bound genes, including MYC and its downstream target genes, were transcriptionally upregulated upon JQ1 treatment. Using BRD2-enriched sites and transcriptional activity analysis, we identified candidate transcription factors potentially involved in the JQ1 response in BRD2-dependent and -independent manner.

Cell-free synthesis of functional antibody fragments to provide a structural basis for antibody-antigen interaction.

Growing numbers of therapeutic antibodies offer excellent treatment strategies for many diseases. Elucidation of the interaction between a potential therapeutic antibody and its target protein by structural analysis reveals the mechanism of action and offers useful information for developing rational antibody designs for improved affinity. Here, we developed a rapid, high-yield cell-free system using dialysis mode to synthesize antibody fragments for the structural analysis of antibody-antigen complexes. Optimal synthesis conditions of fragments (Fv and Fab) of the anti-EGFR antibody 059-152 were rapidly determined in a day by using a 30-µl-scale unit. The concentration of supplemented disulfide isomerase, DsbC, was critical to obtaining soluble antibody fragments. The optimal conditions were directly applicable to a 9-ml-scale reaction, with linear scalable yields of more than 1 mg/ml. Analyses of purified 059-152-Fv and Fab showed that the cell-free synthesized antibody fragments were disulfide-bridged, with antigen binding activity comparable to that of clinical antibodies. Examination of the crystal structure of cell-free synthesized 059-152-Fv in complex with the extracellular domain of human EGFR revealed that the epitope of 059-152-Fv broadly covers the EGF binding surface on domain III, including residues that formed critical hydrogen bonds with EGF (Asp355EGFR, Gln384EGFR, H409EGFR, and Lys465EGFR), so that the antibody inhibited EGFR activation. We further demonstrated the application of the cell-free system to site-specific integration of non-natural amino acids for antibody engineering, which would expand the availability of therapeutic antibodies based on structural information and rational design. This cell-free system could be an ideal antibody-fragment production platform for functional and structural analysis of potential therapeutic antibodies and for engineered antibody development.