Translation Initiation Factor eIF Research Articles

In silico identification and characterisation of pathogenic genetic variants (nsSNPs) in the eukaryotic initiation factors eIF2 and eIF2B affecting miRNA binding sites

BackgroundThe eukaryotic translation initiation factors (eIF2 and eIF2B) are known to play a regulatory role in translation initiation. Studies have indicated that several missense mutations in both eIF2 and eIF2B subunits can lead to severe neurological diseases and cancer. In the current study, we have attempted to identify and characterise the single-nucleotide polymorphisms (SNPs) in the said subunits and their correlation with various diseases.ResultsInterestingly, we could identify SNPs only in 3′ untranslated regions (3′UTR) from EIF2 (EIF2S1 and S3) and EIF2B (EIF2B1, B2 and B5 subunits). Of which, two SNPs, one in each EIF2B1 (rs1050448) and EIF2B2 (rs4556), are observed to be affecting miRNA binding sites. The gene ontology (GO) analysis of identified miRNAs indicates their association with central nervous system development, various stress responses, growth factors, and immune system signalling pathways. Furthermore, molecular docking studies also confirm that the identified miRNAs have an excellent binding ability with corresponding wild-type/mutant dsDNA and mRNA with HADDOCK binding scores in the range of − 38.78 to − 3.99 kcal/mol and − 86.47 to − 23.78 kcal/mol, respectively.ConclusionWe conclude that the identified miRNAs may play a regulatory role in the symptomatic progression of neurological disorders and cancer and the same is validated by existing experimental evidences. Overall, the identified miRNAs serve as potential candidates for carrying out clinical investigations.Graphical abstract

Proteomic Profiling of Unannotated Microproteins in Human Placenta Reveals XRCC6P1 as a Potential Negative Regulator of Translation.

Ribosome profiling and mass spectrometry have revealed thousands of previously unannotated small and alternative open reading frames (sm/alt-ORFs) that are translated into micro/alt-proteins in mammalian cells. However, their prevalence across human tissues and biological roles remains largely undefined. The placenta is an ideal model for identifying unannotated microproteins and alt-proteins due to its considerable protein diversity that is required to sustain fetal development during pregnancy. Here, we profiled unannotated microproteins and alt-proteins in human placental tissues from preeclampsia patients or healthy individuals by proteomics, identified 52 unannotated microproteins or alt-proteins, and demonstrated that five microproteins can be translated from overexpression constructs in a heterologous cell line, although several are unstable. We further demonstrated that one microprotein, XRCC6P1, associates with translation initiation factor eIF3 and negatively regulates translation when exogenously overexpressed. Thus, we revealed a hidden sm/alt-ORF-encoded proteome in the human placenta, which may advance the mechanism studies for placenta development as well as placental disorders such as preeclampsia.

Adaptation of turnip mosaic virus to Arabidopsis thaliana involves rewiring of VPg-host proteome interactions.

The outcome of a viral infection depends on a complex interplay between the host physiology and the virus, mediated through numerous protein-protein interactions. In a previous study, we used high-throughput yeast two-hybrid (HT-Y2H) to identify proteins in Arabidopsis thaliana that bind to the proteins encoded by the turnip mosaic virus (TuMV) genome. Furthermore, after experimental evolution of TuMV lineages in plants with mutations in defense-related or proviral genes, most mutations observed in the evolved viruses affected the VPg cistron. Among these mutations, D113G was a convergent mutation selected in many lineages across different plant genotypes, including cpr5-2 with constitutive expression of systemic acquired resistance. In contrast, mutation R118H specifically emerged in the jin1 mutant with affected jasmonate signaling. Using the HT-Y2H system, we analyzed the impact of these two mutations on VPg's interaction with plant proteins. Interestingly, both mutations severely compromised the interaction of VPg with the translation initiation factor eIF(iso)4E, a crucial interactor for potyvirus infection. Moreover, mutation D113G, but not R118H, adversely affected the interaction with RHD1, a zinc-finger homeodomain transcription factor involved in regulating DNA demethylation. Our results suggest that RHD1 enhances plant tolerance to TuMV infection. We also discuss our findings in a broad virus evolution context.

Correction: The chaperonin CCT interacts with and mediates the correct folding and activity of three subunits of translation initiation factor eIF3: b, i and h.

Correction: The chaperonin CCT interacts with and mediates the correct folding and activity of three subunits of translation initiation factor eIF3: b, i and h.

In silico Characterization of an Initiation Factor 2 Kinase of Black Fungi: A Potential Drug Target for Mycosis

: Fungi infections are responsible for more than 1.6 million deaths per year worldwide. Treatment is time-consuming, compromising the kidney and liver functions. in silico analyses have facilitated the discovery of new drugs that may present fewer side effects. In this connection, kinases that phosphorylate the translation initiation factor eIF-2 are candidate proteins for potent new drugs, which have been recognized as important in maintaining protein synthesis. Substances that interfere with the phosphorylation of the eIF2α factor may be the way to inhibit the production of proteins and accelerate the fungi's death. To determine whether this enzyme can be used as a new drug target, this study aimed to perform in silico functional annotation and characterization of eIF2 factor kinase´s three-dimensional structure from three species of black fungi. In addition, inhibitors that could interact and bind to the active site of the enzyme were explored. The hypothetical protein was submitted to the databases and bioinformatics tools for its characterization, whose analysis of protein-protein interactions was modeled and inhibitors anchored. Protein interaction analysis linked the kinases with other molecules in protein translation and ribosome recycling. However, centrality analysis showed only one kinase as a possible drug target. The inhibitors showed coupling with the active site of protein kinases, and these results indicate a possible blockade of the enzymatic function that can accelerate the response to the drugs. This study demonstrates that biochemical characterization and in silico validation studies of potential drugs can be more efficient and yield faster results.

Nuclear Hsp104 safeguards the dormant translation machinery during quiescence

The resilience of cellular proteostasis declines with age, which drives protein aggregation and compromises viability. The nucleus has emerged as a key quality control compartment that handles misfolded proteins produced by the cytosolic protein biosynthesis system. Here, we find that age-associated metabolic cues target the yeast protein disaggregase Hsp104 to the nucleus to maintain a functional nuclear proteome during quiescence. The switch to respiratory metabolism and the accompanying decrease in translation rates direct cytosolic Hsp104 to the nucleus to interact with latent translation initiation factor eIF2 and to suppress protein aggregation. Hindering Hsp104 from entering the nucleus in quiescent cells results in delayed re-entry into the cell cycle due to compromised resumption of protein synthesis. In sum, we report that cytosolic-nuclear partitioning of the Hsp104 disaggregase is a critical mechanism to protect the latent protein synthesis machinery during quiescence in yeast, ensuring the rapid restart of translation once nutrients are replenished.

The role of protein kinase R in dystonia

Dystonia is a progressive neurological motor disease with few treatment options and no cure. This review synthesizes the results of recent studies that implicate protein kinase R in mediating the molecular mechanisms of dystonia pathogenesis. Mutations in the PKR gene EIF2AK2 and the PKR activator protein PACT are associated with early-onset generalized dystonia. Protein kinase R (PKR) is important for neuronal function. Genetic depletion or inhibition of PKR is associated with increased long-term potentiation and memory, while also causing neuronal hyper-excitability and seizures in mouse models. PKR also senses double stranded RNA within cells and activates the integrated stress response (ISR). The ISR is a conserved signaling pathway that hinges on controlled translational suppression to remodel gene expression during stress. When PKR is activated through binding double stranded RNA or the PKR activator protein PACT, PKR dimerizes, autophosphorylates, and phosphorylates the translation initiation factor eIF2. Translation suppression by p-eIF2 causes stress granule formation and the upregulation of stress-induced genes. The ISR is thought to drive cellular resilience during acute stress. However, chronic ISR activation is associated with neurological diseases, traumatic brain injury, and aging. Neurodevelopmental and neurodegenerative diseases are associated with mutations in other integrated stress response genes, suggesting a critical role for ISR regulation in neuronal health. A growing body of work suggests the ISR is also dysfunctional in dystonia. Future research investigating the molecular mechanisms of the ISR in dystonia will likely reveal therapeutic targets and treatment strategies for this currently incurable disease.

Effect of Gene Silencing of Translation Initiation Factors eIF(iso)4G and eIF(iso)4E on Sour Cherry Rootstock Resistance to Sharka Disease.

Sharka disease, caused by the Plum pox virus (PPV), is one of the most harmful, quarantine viral diseases that affect stone fruit crops. The absence of natural resistance to the virus in stone fruits has become a decisive factor for the use of genetic transformation methods to obtain stable forms. The eIF(iso)4G and eIF(iso)4E genes encode translation initiation factors used in the PPV life cycle. In the presented study, the effect of silencing these genes using the RNA interference method on the resistance of sour cherry rootstock 146-2 plants (Prunus pumila L. x Prunus tomentosa Thunb) to the sharka disease was studied. Two vectors have been created for the genetic transformation of plants, with self-complementary sequences of the eIF(iso)4G and eIF(iso)4E gene fragments. The hairpin expression cassette contains a strong promoter of the peach ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo) gene, as well as an intron and terminator of the same gene. We used the pMF1 vector containing recombinase R and a codA-nptII gene which makes it possible to obtain intragenic marker-free plants. A successful genetic transformation was carried out by the AGL0 strain of A. tumefaciens. Whole leaves of shoots cultivated in vitro were used as a source of explants. Eight independent transgenic lines of rootstock 146-2 were obtained in experiments (sixlines with a hairpin to the eIF(iso)4G gene and two lines with a hairpin to the eIF(iso)4E gene). Their status was confirmed by the PCR and Southern blotting. The obtained plants were acclimatized in a greenhouse. The silencing of the eIF(iso)4G and eIF(iso)4E genes in transgenic plants was confirmed by the quantitative PCR. The presence of specific small interfering (si) RNAs was confirmed by the method of Northern blotting. Plants of all transgenic rootstock lines were infected with PPV by the method of grafting with infected buds. Resistance to the PPV infection of the obtained transgenic plants was carried out by using an enzyme immunoassay. The ELISA results showed that silencing the eIF(iso)4G gene did not lead to increased resistance while silencing the eIF(iso)4E factor gene led to increased resistance to the PPV, and the one line's plants showed no signs of infection for two years after infecting. The work demonstrates a (promising) approach in which the creation of stone cultures resistant to the plum pox virus can be achieved by suppressing the genes of translation initiation factors in clonal rootstocks.

Endoplasmic reticulum oxidoreductin 1-alpha deficiency and activation of protein translation synergistically impair breast tumour resilience.

Endoplasmic reticulum (ER) stress triggers an adaptive response in tumours which fosters cell survival and resilience to stress. Activation of the ER stress response, through its PERK branch, promotes phosphorylation of the α-subunit of the translation initiation factor eIF2, thereby repressing general protein translation and augmenting the translation of ATF4 with the downstream CHOP transcription factor and the protein disulfide oxidase, ERO1-alpha EXPERIMENTAL APPROACH: Here, we show that ISRIB, a small molecule that inhibits the action of phosphorylated eIF2alpha, activating protein translation, synergistically interacts with the genetic deficiency of protein disulfide oxidase ERO1-alpha, enfeebling breast tumour growth and spread. ISRIB represses the CHOP signal, but does not inhibit ERO1. Mechanistically, ISRIB increases the ER protein load with a marked perturbing effect on ERO1-deficient triple-negative breast cancer cells, which display impaired proteostasis and have adapted to a low client protein load in hypoxia, and ERO1 deficiency impairs VEGF-dependent angiogenesis. ERO1-deficient triple-negative breast cancer xenografts have an augmented ER stress response and its PERK branch. ISRIB acts synergistically with ERO1 deficiency, inhibiting the growth of triple-negative breast cancer xenografts by impairing proliferation and angiogenesis. These results demonstrate that ISRIB together with ERO1 deficiency synergistically shatter the PERK-dependent adaptive ER stress response, by restarting protein synthesis in the setting of impaired proteostasis, finally promoting tumour cytotoxicity. Our findings suggest two surprising features in breast tumours: ERO1 is not regulated via CHOP under hypoxic conditions, and ISRIB offers a therapeutic option to efficiently inhibit tumour progression in conditions of impaired proteostasis.

Abstract 3040: The eIF2 kinase GCN2 controls expression of key amino acid transporters and is critical for prostate cancer growth and progression

Abstract The Integrated Stress Response (ISR) is central for adaptation and survival of tumor cells in response to exogenous and endogenous stresses. The ISR features a family of protein kinases that phosphorylate the eukaryotic translation initiation factor eIF2 in response to distinct stresses. While phosphoryation of eIF2 reduces global protein synthesis, there is paradoxical induced translation of select stress adapative gene transcripts, including ATF4, which is central for ISR-directed gene transcription. Therefore, the ISR directs translational and transcriptional control that is critical for cancer adaptation to stress. Recently, eIF2 phosphorylation and ATF4 were suggested to play a role in prostate cancer (PCa) growth and survival; however, the specific function of ISR kinase(s), its mode of activation, and mechanisms by which the ISR facilitate PCa progression are not known.We discovered that the eIF2 kinase GCN2 (EIF2AK4) is activated in a range of PCa cell lines, contributing to enhanced eIF2 phosphorylation and ATF4 expression. Genetic or pharmacological inhibition of GCN2 reduced growth in androgen-sensitive and castration-resistant PCa cell lines in culture and cell line-derived and patient-derived xenograft mouse models in vivo. Induction of GCN2 is accompanied by limitations of select amino acids and accumulation of cognate tRNAs that are reported to be activators of GCN2. A transcriptome analysis of PCa cells treated with a specific small molecular inhibitor of GCN2 indicates that this eIF2 kinase was critical for expression of multiple SLC genes, including amino acid transporters previously implicated in different cancers. We determined that GCN2 inhibition decreases intracellular amino acid levels, and reduced aminoacylation of select tRNAs, accounting for reduced growth in PCa cells. Using CRISPR-based phenotypic screens and genome-wide gene expression analyses of control and GCN2-depleted PCa cells, we confirmed the importance of the transporter genes in PCa fitness. One transporter, SLC3A2 (4F2), is a key SLC gene induced by GCN2 and is essential for PCa proliferation. SLC3A2 has been reported to associate with many SLC7 family transporters, allowing for the localization of these transporters to the plasma membrane for amino acid uptake. Of importance, expression of SLC3A2 partially restored amino acid levels and growth due to loss of GCN2. Our results indicate that select amino acid limitations activate GCN2 in PCa, resulting in the upregulation of key amino acid transporters, including SLC3A2, which provides for nutrient import to facilitate protein synthesis and metabolism required for PCa progression. We conclude that GCN2 and the ISR are promising new therapeutic targets for the treatment of PCa. Citation Format: Ricardo Cordova, Jagannath Misra, Nur P. Damayanti, Parth H. Amin, Kenneth R. Carlson, Angela J. Klunk, Emily T. Mirek, Marcus J. Miller, Tracy G. Anthony, Roberto Pili, Ronald C. Wek, Kirk A. Staschke. The eIF2 kinase GCN2 controls expression of key amino acid transporters and is critical for prostate cancer growth and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3040.

The integrated stress response in the induction of mutant KRAS lung carcinogenesis: Mechanistic insights and therapeutic implications.

The integrated stress response (ISR) is a key determinant of tumorigenesis in response to oncogenic forms of stress like genotoxic, proteotoxic and metabolic stress. ISR relies on the phosphorylation of the translation initiation factor eIF2 to promote the translational and transcriptional reprogramming of gene expression in stressed cells. While ISR promotes tumor survival under stress, its hyperactivation above a level of tolerance can also cause tumor death. The tumorigenic function of ISR has been recently demonstrated for lung adenocarcinomas (LUAD) with KRAS mutations. ISR mediates the translational repression of the dual-specificity phosphatase DUSP6 to stimulate ERK activity and LUAD growth. The significance of this finding is highlighted by the strong anti-tumor responses of ISR inhibitors in pre-clinical LUAD models. Elucidation of the mechanisms of ISR action in LUAD progression via cell-autonomous and immune regulatory mechanisms will provide a better understanding of its tumorigenic role to fully exploit its therapeutic potential in the treatment of a deadly form of cancer.

Targeting GCN2 Regulation of Amino Acid Homeostasis in Prostate Cancer

The Integrated Stress Response (ISR) plays a critical role in the adaptation and survival of tumor cells to exogenous and endogenous stresses. The ISR features four protein kinases (PERK, GCN2, PKR, and HRI), each activated by different stresses, that phosphorylate the eukaryotic translation initiation factor eIF2, resulting in repression of global protein synthesis. Paradoxically, eIF2 phosphorylation also enhances translation of select gene transcripts, including the transcription factor ATF4, which is central for ISR‐directed gene transcription. Therefore, the ISR directs translational and transcriptional control that is critical for cancer stress adaptation. Moreover, eIF2 phosphorylation and ATF4 have recently been suggested to play a role in prostate cancer (PCa) growth and survival; however, the specific function of ISR kinases, their mode of activation, and the mechanisms by which the ISR facilitate PCa progression are unknown.We discovered that GCN2 is activated in a range of PCa cell lines, contributing to enhanced eIF2 phosphorylation and ATF4 expression. Genetic or pharmacological inhibition of GCN2 reduces growth in androgen‐sensitive and castration‐resistant PCa cell lines in culture and cell line‐derived and patient‐derived xenograft mouse models in vivo. Induction of GCN2 is accompanied by limitations of select amino acids and accumulation of cognate tRNAs that are reported to be activators of GCN2.A transcriptome analysis of PCa cells treated with a specific GCN2 small molecular inhibitor indicates that GCN2 is critical for expression of SLCgenes involved in metabolite transport. We found that GCN2 inhibition decreases intracellular amino acid levels accounting for reduced growth in PCa cells. Using CRISPR‐based phenotypic screens and genome‐wide gene expression analyses of wild‐type and GCN2‐depleted PCa cells, we confirmed the importance of the transporter genes in PCa fitness. One transporter, SLC3A2 (4F2), is induced by GCN2 and is essential for PCa proliferation. SLC3A2 engages with many nutrient transporters, allowing for their localization to the plasma membrane. Importantly, expression of SLC3A2 reduced GCN2 activation and rescued decreased amino acid levels and growth inhibition due to loss of GCN2.Our results indicate that select amino acid limitations activate GCN2 in PCa, resulting in the upregulation of key amino acid transporters, including 4F2 (SLC3A2), which provide for nutrient import to facilitate protein synthesis and metabolism required for PCa progression. We conclude that GCN2 and the ISR are promising therapeutic targets for both androgen‐sensitive and castration‐resistant prostate cancer.

A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulation.

In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Previously we showed that translational control is primarily exerted through a conformational switch in eIF2's nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2 (Schoof et al. 2021). Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B's β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed A/I-State model of allosteric ISR regulation.

Proteomic Shifts Reflecting Oxidative Stress and Reduced Capacity for Protein Synthesis, and Alterations to Mitochondrial Membranes in Neurospora crassa Lacking VDAC.

Voltage-dependent anion-selective channels (VDAC) maintain the bidirectional flow of small metabolites across the mitochondrial outer membrane and participate in the regulation of multiple cellular processes. To understand the roles of VDAC in cellular homeostasis, preliminary proteomic analyses of S100 cytosolic and mitochondria-enriched fractions from a VDAC-less Neurospora crassa strain (ΔPor-1) were performed. In the variant cells, less abundant proteins include subunits of translation initiation factor eIF-2, enzymes in the shikimate pathway leading to precursors of aromatic amino acids, and enzymes involved in sulfate assimilation and in the synthesis of methionine, cysteine, alanine, serine, and threonine. In contrast, some of the more abundant proteins are involved in electron flow, such as the α subunit of the electron transfer flavoprotein and lactate dehydrogenase, which is involved in one pathway leading to pyruvate synthesis. Increased levels of catalase and catalase activity support predicted increased levels of oxidative stress in ΔPor-1 cells, and higher levels of protein disulfide isomerase suggest activation of the unfolded protein response in the endoplasmic reticulum. ΔPor-1 cells are cold-sensitive, which led us to investigate the impact of the absence of VDAC on several mitochondrial membrane characteristics. Mitochondrial membranes in ΔPor-1 are more fluid than those of wild-type cells, the ratio of C18:1 to C18:3n3 acyl chains is reduced, and ergosterol levels are lower. In summary, these initial results indicate that VDAC-less N. crassa cells are characterized by a lower abundance of proteins involved in amino acid and protein synthesis and by increases in some associated with pyruvate metabolism and stress responses. Membrane lipids and hyphal morphology are also impacted by the absence of VDAC.

Thermofluor-Based Analysis of Protein Integrity and Ligand Interactions.

Thermofluor is a fluorescence-based thermal shift assay, which measures temperature-induced protein unfolding and thereby yields valuable information about the integrity of a purified recombinant protein. Analysis of ligand binding to a protein is another popular application of this assay. Thermofluor requires neither protein labeling nor highly specialized equipment, and can be performed in a regular real-time PCR instrument. Thus, for a typical molecular biology laboratory, Thermofluor is a convenient method for the routine assessment of protein quality. Here, we provide Thermofluor protocols using the example of Cdc123. This ATP-grasp protein is an essential assembly chaperone of the eukaryotic translation initiation factor eIF2. We also report on a destabilized mutant protein version and on the ATP-mediated thermal stabilization of wild-type Cdc123 illustrating protein integrity assessment and ligand binding analysis as two major applications of the Thermofluor assay.

Multiplexed Analysis of Human uORF Regulatory Functions During the ISR Using PoLib-Seq.

Protein synthesis is a highly regulated essential process. As such, it is subjected to substantial regulation in response to stress. One hallmark of the Integrated Stress Response (ISR) is the immediate shutdown of most translation through phosphorylation of the alpha subunit of translation initiation factor eIF2 and activation of eIF4E binding proteins. While these posttranslational modifications largely inhibit cap-dependent translation, many mRNA resist this inhibition by alternative translation mechanisms involving cis-regulatory sequences and structures in 5' transcript leaders, including upstream Open Reading Frames (uORFs), Internal Ribosome Entry Sites (IRESes), and Cap-Independent Translation Elements (CITEs). Studies of uORF and IRES activity are often performed on a gene-by-gene basis; however, high-throughput methods have recently emerged. Here, we describe a protocol for Polysome Library Sequencing (PoLib-Seq; Fig. 1), a multiplexed assay of reporter gene translation that can be used during the ISR. A designer library of reporter RNAs are transfected into tissue-culture cells, and their translation is assayed via sucrose gradient fractionation followed by high-throughput sequencing. As an example, we include PoLib-seq results simultaneously assaying translation of wildtype and uORF mutant human ATF4 reporter RNAs, recapitulating the known function of uORF1 in resisting translational inhibition during the ISR.

Robust T cell activation requires an eIF3-driven burst in T cell receptor translation.

Activation of T cells requires a rapid surge in cellular protein synthesis. However, the role of translation initiation in the early induction of specific genes remains unclear. Here, we show human translation initiation factor eIF3 interacts with select immune system related mRNAs including those encoding the T cell receptor (TCR) subunits TCRA and TCRB. Binding of eIF3 to the TCRA and TCRB mRNA 3'-untranslated regions (3'-UTRs) depends on CD28 coreceptor signaling and regulates a burst in TCR translation required for robust T cell activation. Use of the TCRA or TCRB 3'-UTRs to control expression of an anti-CD19 chimeric antigen receptor (CAR) improves the ability of CAR-T cells to kill tumor cells in vitro. These results identify a new mechanism of eIF3-mediated translation control that can aid T cell engineering for immunotherapy applications.

Viral evasion of the integrated stress response through antagonism of eIF2-P binding to eIF2B

Viral infection triggers activation of the integrated stress response (ISR). In response to viral double-stranded RNA (dsRNA), RNA-activated protein kinase (PKR) phosphorylates the translation initiation factor eIF2, converting it from a translation initiator into a potent translation inhibitor and this restricts the synthesis of viral proteins. Phosphorylated eIF2 (eIF2-P) inhibits translation by binding to eIF2’s dedicated, heterodecameric nucleotide exchange factor eIF2B and conformationally inactivating it. We show that the NSs protein of Sandfly Fever Sicilian virus (SFSV) allows the virus to evade the ISR. Mechanistically, NSs tightly binds to eIF2B (KD = 30 nM), blocks eIF2-P binding, and rescues eIF2B GEF activity. Cryo-EM structures demonstrate that SFSV NSs and eIF2-P directly compete, with the primary NSs contacts to eIF2Bα mediated by five ‘aromatic fingers’. NSs binding preserves eIF2B activity by maintaining eIF2B’s conformation in its active A-State.

A Multi-Perspective Proximity View on the Dynamic Head Region of the Ribosomal 40S Subunit.

A comparison of overlapping proximity captures at the head region of the ribosomal 40S subunit (hr40S) in Saccharomyces cerevisiae from four adjacent perspectives, namely Asc1/RACK1, Rps2/uS5, Rps3/uS3, and Rps20/uS10, corroborates dynamic co-localization of proteins that control activity and fate of both ribosomes and mRNA. Co-locating factors that associate with the hr40S are involved in (i) (de)ubiquitination of ribosomal proteins (Hel2, Bre5-Ubp3), (ii) clamping of inactive ribosomal subunits (Stm1), (iii) mRNA surveillance and vesicular transport (Smy2, Syh1), (iv) degradation of mRNA (endo- and exonucleases Ypl199c and Xrn1, respectively), (v) autophagy (Psp2, Vps30, Ykt6), and (vi) kinase signaling (Ste20). Additionally, they must be harmonized with translation initiation factors (eIF3, cap-binding protein Cdc33, eIF2A) and mRNA-binding/ribosome-charging proteins (Scp160, Sro9). The Rps/uS-BioID perspectives revealed substantial Asc1/RACK1-dependent hr40S configuration indicating a function of the β-propeller in context-specific spatial organization of this microenvironment. Toward resolving context-specific constellations, a Split-TurboID analysis emphasized the ubiquitin-associated factors Def1 and Lsm12 as neighbors of Bre5 at hr40S. These shuttling proteins indicate a common regulatory axis for the fate of polymerizing machineries for the biosynthesis of proteins in the cytoplasm and RNA/DNA in the nucleus.

EIF3j facilitates loading of release factors into the ribosome.

eIF3j is one of the eukaryotic translation factors originally reported as the labile subunit of the eukaryotic translation initiation factor eIF3. The yeast homolog of this protein, Hcr1, has been implicated in stringent AUG recognition as well as in controlling translation termination and stop codon readthrough. Using a reconstituted mammalian in vitro translation system, we showed that the human protein eIF3j is also important for translation termination. We showed that eIF3j stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the initiation factor eIF3, which also stimulates peptide release, eIF3j activity in translation termination increases. We found that eIF3j interacts with the pre-termination ribosomal complex, and eRF3 destabilises this interaction. In the solution, these proteins bind to each other and to other participants of translation termination, eRF1 and PABP, in the presence of GTP. Using a toe-printing assay, we determined the stage at which eIF3j functions – binding of release factors to the A-site of the ribosome before GTP hydrolysis. Based on these data, we assumed that human eIF3j is involved in the regulation of translation termination by loading release factors into the ribosome.