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Proteins are considered the “workhorses” of the cell and they are produced according tothe central dogma of biology. The general rule is that the genetic information hard-wired intoDNA is transcribed into a messenger RNA (mRNA) molecule which contains the program forprotein synthesis through the translation process. The regulation of protein production canhappen in many ways. The process of mRNA production (transcriptional regulation) has beenstudied extensively and we have a good understanding of how it works. More recently, otherforms of regulation have been gaining attention, particularly translation initiation regulation. Thisis the rate limiting step during translation and it is an important gatekeeper of protein synthesis.This regulation occurs by both the cis-regulatory elements, which are located in the 5′- and 3′-UTRs (untranslated regions), and by trans-acting factors. Translational control of mRNAprovides the cell with a rapid way to control changes in protein concentration and thus acts toassist in maintaining homeostasis while also having a role in modulating more persistentphysiological changes towards cell fate (Sonnenberg and Hinnebusch, 2009). A large proportionof the energy budget of a living cell is funneled into protein synthesis making it intimatelyintegrated with cell metabolism. For this reason, misregulation of translation results inaberrations and several disease phenotypes (Silvera et al., 2010). It is therefore of great value tounderstand detailed aspects of translational control when studying cell homeostasis and disease.Untranslated regions of messenger RNAs are populated with a variety of regulatory structuressuch as stem-loop structures, upstream initiation codons and open reading frames, internalribosome entry sites and cis-acting elements that interact with RNA-binding proteins. In thepresent work, I will discuss the importance of untranslated elements on the 5’ and the 3’ ends ofspecific transcripts and how interactions with these regions alter the interplay between the RNAand the translation machinery, focusing on eukaryotic translation initiation factor eIF3,the largesttranslation initiation factor.

Cisplatin (CDDP)-based chemotherapy is the first-line treatment for muscle-invasive and metastatic bladder cancer, yet most patients rapidly develop resistance. N6-methyladenosine (m6A) methylation is a pervasive RNA modification, and its specific role and potential mechanism in the regulation of CDDP chemosensitivity in bladder cancer remain unclear. Furthermore, studies have not yet fully elucidated whether circular RNA (circRNA) can directly regulate m6A modification of mRNA. Here we report upregulation of a novel circRNA, hsa_circ_0008399 (circ0008399), by eukaryotic translation initiation factor 4A3 (EIF4A3) in bladder cancer tissues and cell lines. Functionally, circ0008399 inhibited apoptosis of bladder cancer cells. Mechanistically, circ0008399 bound Wilms' tumor 1–associating protein (WTAP) to promote formation of the WTAP/METTL3/METTL14 m6A methyltransferase complex. Circ0008399 increased expression of TNF alpha-induced protein 3 (TNFAIP3) by increasing its mRNA stability in an m6A-dependent manner. In patients with bladder cancer, high expression of circ0008399 and WTAP was associated with poor outcomes. Importantly, activation of the circ0008399/WTAP/TNFAIP3 pathway decreased bladder cancer chemosensitivity to CDDP, and targeting the circ0008399/WTAP/TNFAIP3 axis enhanced the CDDP efficacy. Collectively, these findings give novel insights into circRNA-mediated regulation of m6A modifications and provide potential therapeutic targets for bladder cancer. Significance: A newly characterized circRNA circ0008399 binds WTAP to modulate expression of target RNA through m6A modification and reduce cisplatin sensitivity in bladder cancer, implicating the potential therapeutic value of targeting this axis.

Breast cancer (BC) is one of the most common malignancies among women worldwide with a high incidence of recurrence and metastasis. In this study, we demonstrate that hsa_circ_0068631, a circRNA generated from the transferrin receptor (TFRC), is upregulated in BC tissues and cell lines. Knockdown of hsa_circ_0068631 inhibited the proliferation and migration of BC cells in vitro and in vivo. Mechanistically, an RNA pull-down assay and RNA immunoprecipitation assay revealed that eukaryotic translation initiation factor 4A3 (EIF4A3) could bind to hsa_circ_0068631 and c-Myc mRNA. Additionally, the expression of hsa_circ_0068631 was positively correlated with c-Myc, and the upregulation of hsa_circ_0068631 was a crucial factor for the dysregulation of c-Myc. Through an actinomycin D assay, we confirmed that the mRNA stability of c-Myc was influenced by hsa_circ_0068631 and EIF4A3. Furthermore, hsa_circ_0068631 could recruit EIF4A3 to increase c-Myc mRNA stability. Rescue assays manifesting depletion of c-Myc rescued the promotive effect of hsa_circ_0068631 overexpression on biological activities in BC. In conclusion, to our knowledge, this study is the first to unveil the role of hsa_circ_0068631 and the hsa_circ_0068631/EIF4A3/c-Myc axis in BC, providing a new target for BC treatment., Graphical abstract, circRNA hsa_circ_0068631 was upregulated in BC tissues and cell lines. hsa_circ_0068631 promoted the proliferation and migration of BC cells in vitro and in vivo. Mechanistically, hsa_circ_0068631 could recruit EIF4A3 to increase c-Myc mRNA stability, unveiling the role of the hsa_circ_0068631/EIF4A3/c-Myc axis in BC.

GGGGCC (G4C2) repeat expansion in the first intron of C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-containing RNA is translated into dipeptide repeat (DPR) proteins, some of which are neurotoxic. Using dynamic ribosome profiling, we identified three translation initiation sites in the intron upstream of (G4C2) repeats; these sites are detected irrespective of the presence or absence of the repeats. During translocation, ribosomes appear to be stalled on the repeats. An AUG in the preceding C9ORF72 exon initiates a uORF that inhibits downstream translation. Polysome isolation indicates that unspliced (G4C2) repeat-containing RNA is a substrate for DPR protein synthesis. (G4C2) repeat-containing RNA translation is 5′ cap-independent but inhibited by the initiation factor DAP5, suggesting an interplay with uORF function. These results define novel translational mechanisms of expanded (G4C2) repeat-containing RNA in disease.

Our work was to evaluate Exosome CircRNA EIF4G2 in cervical cancer development. Methods: Using Hela and Siha in our present study. Transfection vector, exosome cirEIF4G2, exosome si-NC or exosome si-circEIF4G2 in cells. Using RT-qPCR to measure circEIF4G2 gene expression in difference cell groups. Evaluating cell proliferation, apoptosis, invasion and wound healing rate by MTT, flow cytometry, transwell and wound healing assay. The relative proteins, HPV16 E6 and HPV16 E7, were evaluated by WB assay. With Exosome CircRNA EIF4G2 transfection, Hela and Siha cells proliferation, invasion cells number and wound healing rates were significantly increased and cells apoptosis were significantly depressed (P < 0.001, respectively) with HPV16 E6 and HPV16 E7 proteins expression were significantly up-regulation (P < 0.001, respectively). However, with Exosome si-CircRNA EIF4G2 transfection, Hela and Siha cells proliferation, invasion cells number and wound healing rates were significantly depressed and cells apoptosis were significantly increased (P < 0.001, respectively) with HPV16 E6 and HPV16 E7 proteins expression were significantly down-regulation (P < 0.001, respectively). Exosome CircRNA EIF4G2 as an oncology role in cervical cancer via regulation HPV16 E6/E7 up-regulation in vitro study.

Bacterial infections continue to represent a major worldwide health hazard following the emergence of drug-resistant pathogenic strains. Pseudomonas aeruginosa is an opportunistic pathogen causing nosocomial infections with increased morbidity and mortality. The increasing antibiotic resistance in P. aeruginosa has led to an unmet need for discovery of new antibiotic candidates. Bacterial protein synthesis is an essential metabolic process and a validated target for antibiotic development; however, the precise structural mechanism in P. aeruginosa remains unknown. In this work, the interaction of P. aeruginosa initiation factor 1 (IF1) with the 30S ribosomal subunit was studied by NMR, which enabled us to construct a structure of IF1-bound 30S complex. A short α-helix in IF1 was found to be critical for IF1 ribosomal binding and function. A peptide derived from this α-helix was tested and displayed a high ability to inhibit bacterial growth. These results provide a clue for rational design of new antimicrobials.

Preeclampsia (PE) has been recognized as one of the main reasons for neonatal and maternal mortality and morbidity. This study intended to identify certain genes that correlated with the pathogenesis of PE, and disclose the underlying mechanisms. The GSE14776 and GSE65271 datasets were obtained from the Gene Expression Omnibus database. Venn diagram analysis was performed to identify the differently expressed genes. The potential pathways were analyzed by Gene set enrichment analysis software. The expression of eukaryotic translation initiation factor 3 subunit D (EIF3D) in tissues and cells was respectively tested by immunohistochemistry and the quantitative real-time PCR. Cell transfection was utilized to alter the expression of EIF3D. Cell proliferation, invasion and migration were respectively tested by MTT, EdU, transwell and wound healing assays. Tube formation assay was utilized to determine the tube formation capacity of HTR-8/SVneo cells. ELISA was employed for determination of the concentration of Angiotensin (ANG)-1. Moreover, the expression of EIF3D, proliferation-, metastasis-, tube formation- and MAPK/ERK1/2 pathway-related proteins were measured utilizing western blot. EIF3D was selected in this study. EIF3D was upregulated in placentas tissues collected from patients with PE. EIF3D upregulation observably repressed the proliferation, invasion, migration, wound healing and tube formation of HTR-8/SVneo cells, and the expression of their associated proteins. Besides, the concentration of ANG-1, and the ratios of phosphorylated-ERK1/2 and phosphorylated-MEK1/MEK1 were also markedly lowered by EIF3D upregulation. Whereas, EIF3D knockdown exerted the opposite effects, and these effects were distinctly counteracted by ERK1/2 inhibitor SC-221593 treatment. In conclusion, these observations manifested that EIF3D upregulation might have repressed the progression of PE through modulation of MAPK/ERK1/2 pathway.

A hexanucleotide repeat expansion GGGGCC in the non-coding region of C9orf72 is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Toxic dipeptide repeats (DPRs) are synthesized from GGGGCC via repeat-associated non-AUG (RAN) translation. Here, we develop C. elegans models that express, either ubiquitously or exclusively in neurons, 75 GGGGCC repeats flanked by intronic C9orf72 sequence. The worms generate DPRs (poly-glycine-alanine [poly-GA], poly-glycine-proline [poly-GP]) and poly-glycine-arginine [poly-GR]), display neurodegeneration, and exhibit locomotor and lifespan defects. Mutation of a non-canonical translation-initiating codon (CUG) upstream of the repeats selectively reduces poly-GA steady-state levels and ameliorates disease, suggesting poly-GA is pathogenic. Importantly, loss-of-function mutations in the eukaryotic translation initiation factor 2D (eif-2D/eIF2D) reduce poly-GA and poly-GP levels, and increase lifespan in both C. elegans models. Our in vitro studies in mammalian cells yield similar results. Here, we show a conserved role for eif-2D/eIF2D in DPR expression.

Healthy aging requires the coordination of numerous stress signaling pathways that converge on the protein homeostasis network. The integrated stress response (ISR) is activated by diverse stimuli, leading to phosphorylation of the eukaryotic translation initiation factor eIF2 in its α-subunit. Under replete conditions, eIF2 orchestrates 5′ cap–dependent mRNA translation and is thus responsible for general protein synthesis. eIF2α phosphorylation, the key event of the ISR, reduces global mRNA translation while enhancing the expression of a signature set of stress response genes. Despite the critical role of protein quality control in healthy aging and in numerous longevity pathways, the role of the ISR in longevity remains largely unexplored. ISR activity increases with age, suggesting a potential link with the aging process. Although decreased protein biosynthesis, which occurs during ISR activation, has been linked to lifespan extension, recent data show that lifespan is limited by the ISR as its inhibition extends survival in nematodes and enhances cognitive function in aged mice. Here we survey how aging affects the ISR, the role of the ISR in modulating aging, and pharmacological interventions to tune the ISR. Finally, we will explore the ISR as a plausible target for clinical interventions in aging and age-related disease. The integrated stress response governs adaptive reprogramming of protein synthesis following external or internal signals. The authors discuss its role in aging and longevity, and highlight strategies to enhance health and resilience.

Signaling via the B-cell receptor (BCR) is a key driver and therapeutic target in chronic lymphocytic leukemia (CLL). BCR stimulation of CLL cells induces expression of eIF4A, an initiation factor important for translation of multiple oncoproteins, and reduces expression of PDCD4, a natural inhibitor of eIF4A, suggesting that eIF4A may be a critical nexus controlling protein expression downstream of the BCR in these cells. We, therefore, investigated the effect of eIF4A inhibitors (eIF4Ai) on BCR-induced responses. We demonstrated that eIF4Ai (silvestrol and rocaglamide A) reduced anti-IgM-induced global mRNA translation in CLL cells and also inhibited accumulation of MYC and MCL1, key drivers of proliferation and survival, respectively, without effects on upstream signaling responses (ERK1/2 and AKT phosphorylation). Analysis of normal naïve and non-switched memory B cells, likely counterparts of the two main subsets of CLL, demonstrated that basal RNA translation was higher in memory B cells, but was similarly increased and susceptible to eIF4Ai-mediated inhibition in both. We probed the fate of MYC mRNA in eIF4Ai-treated CLL cells and found that eIF4Ai caused a profound accumulation of MYC mRNA in anti-IgM treated cells. This was mediated by MYC mRNA stabilization and was not observed for MCL1 mRNA. Following drug wash-out, MYC mRNA levels declined but without substantial MYC protein accumulation, indicating that stabilized MYC mRNA remained blocked from translation. In conclusion, BCR-induced regulation of eIF4A may be a critical signal-dependent nexus for therapeutic attack in CLL and other B-cell malignancies, especially those dependent on MYC and/or MCL1.

Translation reinitiation is a gene-specific translational control mechanism. It is characterized by the ability of short upstream ORFs to prevent full ribosomal recycling and allow the post-termination 40S subunit to resume traversing downstream for the next initiation event. It is well known that variable transcript-specific features of various uORFs and their prospective interactions with initiation factors lend them an unequivocal regulatory potential. Here, we investigated the proposed role of the major initiation scaffold protein eIF4G in reinitiation and its prospective interactions with uORF’s cis-acting features in yeast. In analogy to the eIF3 complex, we found that eIF4G and eIF4A but not eIF4E (all constituting the eIF4F complex) are preferentially retained on ribosomes elongating and terminating on reinitiation-permissive uORFs. The loss of the eIF4G contact with eIF4A specifically increased this retention and, as a result, increased the efficiency of reinitiation on downstream initiation codons. Combining the eIF4A-binding mutation with that affecting the integrity of the eIF4G1–RNA2-binding domain eliminated this specificity and produced epistatic interaction with a mutation in one specific cis-acting feature. We conclude that similar to humans, eIF4G is retained on ribosomes elongating uORFs to control reinitiation also in yeast.

The protein stability of the initiation factors Orc2, Orc3, Orc4, and Cdc6 was analyzed after UV light exposure in two human cell lines. In the cell line with higher repair capacity, HEK 293, no changes in the cell cycle distribution or in the protein levels of the investigated factors were detected. In HeLa cells that are characterized by lower repair capacity, UV irradiation caused a reduction of the levels of Cdc6, Orc2 and Orc3, but not of Orc4 or triggered apoptosis. The appearance of the truncated 49 kDa form of Cdc6 suggested the involvement of the caspase pathway in the degradation of the proteins. Reduced protein levels of Cdc6 were detected in UV damaged HeLa cells in which the apoptotic process was blocked with the caspase inhibitor Z-VAD-fmk, indicating that the degradation of Cdc6 is mediated by the proteasome pathway instead. In the presence of caffeine, an inhibitor of the cell cycle checkpoint kinases, Cdc6 was stabilized, demonstrating that its degradation is controlled by the DNA damage cell cycle checkpoint. We conclude that in response to DNA damage, the activation of origins of replication can be prevented by the degradation of Cdc6, most likely through the proteasome pathway.

Qianggui He, Lijun Tao, Hongbo Xu, Xianhai Xie, Shuibing Cheng Department of Trauma Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, People’s Republic of ChinaCorrespondence: Lijun TaoDepartment of Trauma Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, People’s Republic of ChinaEmail woodchuck2020@163.comBackground: Hepatocellular carcinoma (HCC) is a major threat for human health. This work aimed to determine the potential function of circ_0072995 in HCC progression and its molecular mechanism.Methods: qRT-PCR was conducted to analyze circ_0072995 expression. CCK8 and colony formation assays were utilized to detect cell proliferation. Transwell assay was performed to determine migration and invasion. Interactions among circ_0072995, miR-1253 and EIF4A3 (Eukaryotic Translation Initiation Factor 4A3) were predicted through bioinformatics methods and confirmed via luciferase reporter assay and RNA pulldown assay.Results: circ_0072995 expression was upregulated in HCC tissues. Circ_0072995 high level was associated with poor prognosis. Circ_0072995 knockdown impaired proliferation, migration, invasion and survival. MiR-1253 was sponged by circ_0072995 and targeted EIF4A3 directly. Circ_0072995 inhibited miR-1253 to upregulate EIF4A3 level.Conclusion: Circ_0072995 exerted tumorigenic roles to enhance HCC progression through activating EIF4A3 by sponging miR-1253.Keywords: circ_0072995, miR-1253, EIF4A3, HCC

Trichogramma chilonis is an important natural enemy for control of various Lepidoperan crop pests. The biology of T. chilonis is well-studied, but the molecular mechanisms of this biology require further study. Screening suitable reference genes is a vital step for use of RT-qPCR to understand underlying molecular physiology. In the present study, nine candidate reference genes including elongation factor 2 (EF2), ribosomal proteins (RPS23, RPL13, and RPL44), malate dehydrogenase (MDH), eukaryotic translation initiation factor 3 subunit F (EIF3F), zinc finger protein 268 (ZFP268), muscle specific protein 20 (MP20), and ATP synthase subunit alpha (ATP5F1A) were evaluated at different conditions including development stage, diet, temperature, and insecticide treatments. Four common algorithms (the Delta Ct method, geNorm, BestKeeper, and NormFinder) and RefFinder were used to analyze gene expression stability. Our results indicated that two reference genes used for normalization were sufficient, and the optimal combinations were: RPS23 and EF2 for developmental stages, ZFP268 and EF2 for feeding with different diets, ZFP268 and RPL13 for temperature treatments, and EF2 and RPL44 for insecticide treatments. The results provide preliminary determination of suitable reference gene for standard RT-qPCR analyses in T. chilonis, which might establish the foundation for further molecular biology research.

The addition of the methyl-7-guanosine (m7G) “cap” on the 5' ends of coding and some non-coding RNAs is essential for their protein coding capacity and biochemical activity, respectively. It was previously considered that capping was a constitutive process that generates a complete cap on all transcripts at steady-state. However, development of new methodologies demonstrated that steady-state capping is a dynamic and regulatable feature of many coding and non-coding RNAs. Indeed, capping status of specific RNAs can flux during differentiation and development, thereby impacting on their protein-coding capacity and activity. Moreover, in some primary cancer specimens, capping can be elevated for transcripts encoding proteins involved in proliferation and survival corresponding to their increased protein levels. Overexpression of one of the capping enzymes (RNMT), the transcription factor MYC or the eukaryotic translation initiation factor eIF4E all led to increased levels of steady-state capping of selected transcripts. Additionally, transcripts can be decapped and recapped, allowing these to be sequestered until needed. This review provides a summary of the major advances in enzymatic and affinity-based approaches to quantify m7G capping. Further, we summarize the evidence for regulation of capping. Capping has emerged as a significant regulatory step in RNA metabolism which is poised to impact a myriad of biological processes.

Newly synthesized mRNA is translated during its export through the nuclear pore complex, when its 5′-cap structure is still bound by the nuclear cap-binding complex (CBC), a heterodimer of cap-binding protein (CBP) 80 and CBP20. Despite its critical role in mRNA surveillance, the mechanism by which CBC-dependent translation (CT) is regulated remains unknown. Here, we demonstrate that the CT initiation factor (CTIF) is tethered in a translationally incompetent manner to the perinuclear region by the DEAD-box helicase 19B (DDX19B). DDX19B hands over CTIF to CBP80, which is associated with the 5′-cap of a newly exported mRNA. The resulting CBP80–CTIF complex then initiates CT in the perinuclear region. We also show that impeding the interaction between CTIF and DDX19B leads to uncontrolled CT throughout the cytosol, consequently dysregulating nonsense-mediated mRNA decay. Altogether, our data provide molecular evidence supporting the importance of tight control of local translation in the perinuclear region.

The sensitivity of pigs to deoxynivalenol (DON) might be increased by systemic inflammation (SI), which also has consequences for hepatic integrity. Liver lesions and a dys-regulated gene network might hamper hepatic handling and elimination of DON whereby the way of initiation of hepatic inflammation might play an additional role. First and second-pass exposure of the liver with LPS for triggering a SI was achieved by LPS infusion via pre- or post-hepatic venous route, respectively. Each infusion group was pre-conditioned either with a control diet (0.12 mg DON/kg diet) or with a DON-contaminated diet (4.59 mg DON/kg diet) for 4 wk. Liver transcriptome was evaluated at 195 min after starting infusions. DON exposure alone failed to modulate the mRNA expression significantly. However, pre- and post-hepatic LPS challenges prompted transcriptional responses in immune and metabolic levels. The mRNAs for B-cell lymphoma 2-like protein 11 as a key factor in apoptosis and IFN-γ released by T cells were clearly up-regulated in DON-fed group infused with LPS post-hepatically. On the other hand, mRNAs for nucleotide binding oligomerization domain containing 2, IFN-α and eukaryotic translation initiation factor 2α kinase 3 as ribosomal stress sensors were exclusively up-regulated in control pigs with pre-hepatic LPS infusion. These diverse effects were traced back to differences in TLR4 signalling.

During autophagy, the coordinated actions of autophagosomes and lysosomes result in the controlled removal of damaged intracellular organelles and superfluous substrates. The evolutionary conservation of this process and its requirement for maintaining cellular homeostasis emphasizes the need to better dissect the pathways governing its molecular regulation. In our previously performed high-content screen, we assessed the effect of 1530 RNA-binding proteins on autophagy. Among the top regulators, we identified the eukaryotic translation initiation factor 4A-3 (eIF4A3). Here we show that depletion of eIF4A3 leads to a potent increase in autophagosome and lysosome biogenesis and an enhanced autophagic flux. This is mediated by the key autophagy transcription factor, TFEB, which becomes dephosphorylated and translocates from the cytoplasm to the nucleus where it elicits an integrated transcriptional response. We further identified an exon-skipping event in the transcript encoding for the direct TFEB kinase, GSK3B, which leads to a reduction in GSK3B expression and activity. Through analysis of TCGA data, we found a significant upregulation of eIF4A3 expression across several cancer types and confirmed the potential relevance of this newly identified signaling axis in human tumors. Hence, our data suggest a previously unrecognized role for eIF4A3 as a gatekeeper of autophagy through the control of TFEB activation, revealing a new mechanism for autophagy regulation.

Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factorσto form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensuslacUV5promoters using high-throughput single-molecule magnetic tweezers. We probed the key protein–DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RPOis a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPOstabilization may involve sequence-independent interactions between the DNA and the holoenzyme, driven by a non-Coulombic effect consistent with the non-template DNA strand interacting withσand the RNAPβsubunit. The temperature dependence provides the energy scale of open-complex formation and further supports the existence of additional intermediates.

12 Pág. Centro de Biotecnología y Genómica de Plantas, Mutations in the eukaryotic translation initiation factors eIF4E and eIF(iso)4E confer potyvirus resistance in a range of plant hosts. This supports the notion that, in addition to their role in translation of cellular mRNAs, eIF4E isoforms are also essential for the potyvirus cycle. CERES is a plant eIF4E- and eIF(iso)4E-binding protein that, through its binding to the eIF4Es, modulates translation initiation; however, its possible role in potyvirus resistance is unknown. In this article, we analyse if the ectopic expression of AtCERES is able to interfere with turnip mosaic virus replication in plants. Our results demonstrate that, during infection, the ectopic expression of CERES in Nicotiana benthamiana promotes the development of a mosaic phenotype when it is accumulated to moderate levels, but induces veinal necrosis when it is accumulated to higher levels. This necrotic process resembles a hypersensitive response (HR)-like response that occurs with different HR hallmarks. Remarkably, Arabidopsis plants inoculated with a virus clone that promotes high expression of CERES do not show signs of infection. These final phenotypical outcomes are independent of the capacity of CERES to bind to eIF4E. All these data suggest that CERES, most likely due to its leucine-rich repeat nature, could act as a resistance protein, able to promote a range of different defence responses when it is highly overexpressed from viral constructs., This research has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC Grant Agreement n. 260468 to MMC and from the grant S2013-ABI2734 from CAM. In addition, this work has been financially supported by RTI2018-095946-B100 from MICIU and by the ‘Severo Ochoa Programme for Centres of Excellence in R&D’ from the Agencia Estatal de Investigación of Spain (grant SEV-2016-0672 [2017–2021]) to the CBGP. In the frame of this latter program, RT was supported with a postdoctoral contract.

SARS-CoV-2 is an enveloped positive-sense RNA virus, contain crown-like spikes on its surface, exceptional of large RNA genome, and a special replication machinery. Common symptoms of SARS-CoV-2 include cough, common cold, fever, sore throat, and a variety of severe acute respiratory disease (SARD) such as pneumonia. SARS-CoV-2 infects epithelial cells, T-cells, macrophages, and dendritic cells and also influences the production and implantation of pro-inflammatory cytokines and chemokines. Repurposing of various drugs during this emergency condition can reduce the rate of mortality as well as time and cost. Two druggable protein and enzyme targets have been selected in this review article due to their crucial role in the viral life cycle. The eukaryotic translation initiation factor (eIF4A), cyclophilin, nucleocapsid protein, spike protein, Angiotensin-converting enzyme 2 (ACE2), 3-chymotrypsin-like cysteine protease (3CLpro), and RNA-dependent RNA polymerase (RdRp) play significant role in early and late phase of SARS-CoV-2 replication and translation. This review paper is based on the rationale of inhibiting of various SARS-CoV-2 proteins and enzymes as novel therapeutic approaches for the management and treatment of patients with SARS-CoV-2 infection. We also discussed the structural and functional relationship of different proteins and enzymes to develop therapeutic approaches for novel coronavirus SARS-CoV-2.

p53 is critical for tumor suppression but also elicits detrimental effects when aberrantly overexpressed. Thus, multiple regulators, including RNA-binding protein RBM38, are found to tightly control p53 expression. Interestingly, RBM38 is unique in that it can either suppress or enhance p53 mRNA translation via altered interaction with eIF4E potentially mediated by serine-195 (S195) in RBM38. Thus, multiple RBM38/eIF4E knock-in (KI) cell lines were generated to investigate the significance of eIF4E-RBM38 interaction in controlling p53 activity. We showed that KI of RBM38-S195D or -Y192C enhances, whereas KI of RBM38-S195K/R/L weakens, the binding of eIF4E to p53 mRNA and subsequently p53 expression. We also showed that KI of eIF4E-D202K weakens the interaction of eIF4E with RBM38 and thereby enhances p53 expression, suggesting that D202 in eIF4E interacts with S195 in RBM38. Moreover, we generated an Rbm38 S193D KI mouse model in which human-equivalent serine-193 is substituted with aspartic acid. We showed that S193D KI enhances p53-dependent cellular senescence and that S193D KI mice have a shortened life span and are prone to spontaneous tumors, chronic inflammation, and liver steatosis. Together, we provide in vivo evidence that the RBM38-eIF4E loop can be explored to fine-tune p53 expression for therapeutic development.

Internal tandem duplication (-ITD) mutations of Fms-like tyrosine kinase 3 (FLT3) provide growth and pro-survival signals in the context of established driver mutations in FLT3 mutant acute myeloid leukemia (AML). Maternal embryonic leucine zipper kinase (MELK) is an aberrantly expressed gene identified as a target in AML. The MELK inhibitor OTS167 induces cell death in AML including cells with FLT3 mutations, yet the role of MELK and mechanisms of OTS167 function are not understood. OTS167 alone or in combination with tyrosine kinase inhibitors (TKIs) were used to investigate the effect of OTS167 on FLT3 signaling and expression in human FLT3 mutant AML cell lines and primary cells. We describe a mechanism whereby OTS167 blocks FLT3 expression by blocking FLT3 translation and inhibiting phosphorylation of eukaryotic initiation factor 4E–binding protein 1 (4E-BP1) and eukaryotic translation initiation factor 4B (eIF4B). OTS167 in combination with TKIs results in synergistic induction of FLT3 mutant cell death in FLT3 mutant cell lines and prolonged survival in a FLT3 mutant AML xenograft mouse model. Our findings suggest signaling through MELK is necessary for the translation and expression of FLT3-ITD, and blocking MELK with OTS167 represents a viable therapeutic strategy for patients with FLT3 mutant AML.

Banana bract mosaic virus (BBrMV), belongs to the genus Potyvirus and it is an important viral pathogen of bananas and plantains. The eukaryotic translation initiation factor, eIF4E, and its isoform play key roles during the virus infection in plants, particularly Potyvirus. The present study was undertaken to determine the role of BBrMV-viral protein genome-linked (VPg) in virus infectivity by analyzing the interaction with the eukaryotic translation initiation factor eIF4E through yeast two-hybrid system. The results suggest that plantain cv. Nendran eIF4E plays an essential role in the initiation of the translation of capped mRNAs and its association with VPg would point to a role of the viral protein in the translation of the virus and may potentially contribute to BBrMV resistance. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13337-021-00672-9) contains supplementary material, which is available to authorized users.

The bipolar spindle structure in meiosis is essential for faithful chromosome segregation. PUTATIVE RECOMBINATION INITIATION DEFECT 1 (PRD1) previously has been shown to participate in the formation of DNA double strand breaks (DSBs). However, the role of PRD1 in meiotic spindle assembly has not been elucidated. Here, we reveal by both genetic analysis and immunostaining technology that PRD1 is involved in spindle assembly in rice (Oryza sativa) meiosis. We show that DSB formation and bipolar spindle assembly are disturbed in prd1 meiocytes. PRD1 signals display a dynamic pattern of localization from covering entire chromosomes at leptotene to congregating at the centromere region after leptotene. Centromeric localization of PRD1 signals depends on the organization of leptotene chromosomes, but not on DSB formation and axis establishment. PRD1 exhibits interaction and co-localization with several kinetochore components. We also find that bi-orientation of sister kinetochores within a univalent induced by mutation of REC8 can restore bipolarity in prd1. Furthermore, PRD1 directly interacts with REC8 and SGO1, suggesting that PRD1 may play a role in regulating the orientation of sister kinetochores. Taken together, we speculate that PRD1 promotes bipolar spindle assembly, presumably by modulating the orientation of sister kinetochores in rice meiosis.

This study aimed to analyze the relative expression of Eukaryotic Translation Initiation Factor 3 Subunit B (EIF3B) in pancreatic cancer and elucidate its contribution to this disease.Relative expression of EIF3B in pancreatic cancer was analyzed by immunohistochemistry. Cell viability was determined by the MTT assay and cell proliferation was measured by direct cell counting. Cell apoptosis was detected by Annexin V staining followed by flow cytometry analysis, and cell cycle was analyzed by PI staining. The differential expression gene analysis was performed by microarray. Tumor progression in response to EIF3B deficiencyWe found aberrantly high expression of EIF3B in pancreatic cancer, which associated with unfavorable prognosis. Knockdown of EIF3B greatly compromised cell viability and proliferation in both SW1990 and PANC-1 cells. Furthermore, EIF3B deficiency induced cell cycle arrest and spontaneous apoptosis.Our data uncovered the oncogenic role of EIF3B in pancreatic cancer.

The plant eukaryotic translation-initiation factors eIF4E and eIF(iso)4E play key roles in infection by plant RNA viruses, especially potyviruses. Mutations in the genes that encode these factors reduce susceptibility to the viruses. In the amphidiploid plant tobacco (Nicotiana tabacum L.), eIF4E1-S deletion mutants resist Potato virus Y (PVY), but resistance-breaking strains (RB-PVY) have appeared. In an earlier study, we demonstrated that the loss-of-function of eIF(iso)4E-T reduces susceptibility to RB-PVY. Here, we show that simultaneous inhibition of eIF4E1-S and eIF(iso)4E-T synergistically confers enhanced resistance to both PVY and RB-PVY without host growth or development defects. PVY symptoms and accumulation in a tobacco line lacking eIF4E1-S were detected at 14 days post-inoculation (dpi) and RB-PVY symptoms in lines without functional eIF(iso)4E-T were observed at 24 dpi. RB-PVY emerged in a PVY-infected tobacco line lacking eIF4E1-S. In contrast, lines without functional eIF4E1-S and eIF(iso)4E-T were nearly immune to PVY and RB-PVY, and little accumulation of either virus was detected even at 56 dpi. Thus, the lines will be promising for PVY-resistance breeding. This study provides a novel strategy to develop tobacco highly resistant to PVY and RB-PVY, and insights into the mechanisms responsible for high-level resistance.

Haloxylon salicornicum is a xero-halophyte which grow predominantly in dry saline areas. However, the proteomic approach for revealing the regulatory network involved in salt adaptation of this important xerohalophyte has not been studied so far. In the present investigation, the label-free quantitative proteomic analysis was carried out in shoot of H. salicornicum to get an insight into the functional network of proteins involved in salt tolerance. Comparative proteomic analysis in control and salt treated plants of H. salicornicum by nano-ESI-LC-MS and MS/MS, and data base searching led to the identification of 723 proteins. Pathway enrichment analysis by KEGG uncovered various biological pathways to which salinity induced differentially regulated proteins are involved. In H. salicornicum, out of 723 identified proteins, 187 proteins were differentially regulated in response to salinity. In addition to significant up-regulation of stress responsive proteins, other proteins involved in carbohydrate metabolism, TCA cycle, protein synthesis, antioxidative defense systems, energy transfer, ion transport, nucleotide binding, and proteosomal proteins also significantly up-regulated under salinity in H. salicornicum. The major photosynthetic proteins up-regulated were RuBisCo, D1 protein, photosystem II-CP47, and cytochrome b599. TCA cycle component proteins such as citrate synthase, succinate dehydrogenase, and malate dehydrogenase upregulated indicating their significant roles in providing vital energy for salinity tolerance. Salinity induced higher expressions of ion transporters in H. salicornicum suggest efficient compartmentalization of toxic sodium ions. In addition, up-regulation of antioxidative defense system can be correlated with effective scavenging of salinity induced ROS, hence imparting salt tolerance. In H. salicornicum, protein synthesis was boosted under salinity as confirmed from the salinity-induced up-regulation of the ribosome associated proteins. Salinity induced significantly changed proteins of the ribosomal pathway include ribosomal protein components such as elongation factor-Tu (EF-Tu), initiation factor 1 and 2 (IF1, 2), Rpo cluster C and B, etc. Functional integrity of protein synthesis machinery in H. salicornicum is maintained under high salinity by higher abundance of ribosomal subunit proteins in NaCl-treated plants. We assume that consistent energy supply by the up-regulation of TCA cycle along with uninterrupted protein synthesis and maintenance of structural integrity of the photosynthetic machinery are the primary mechanism of salinity tolerance of H. salicornicum. In the present study, we comprehensively elucidated possible mechanisms associated with systematic salt tolerance of H. salicornicum employing proteomic approach. The information from this study will contribute to the genetic improvement of crop plants that can be grown in saline marginal lands.

Background The BCL2 inhibitor venetoclax has shown efficacy in several hematologic malignancies, with the greatest response rates in indolent blood cancers such as chronic lymphocytic leukaemia. There is a lower response rate to venetoclax monotherapy in diffuse large B-cell lymphoma (DLBCL). Methods We tested inhibitors of cap-dependent mRNA translation for the ability to sensitise DLBCL and mantle cell lymphoma (MCL) cells to apoptosis by venetoclax. We compared the mTOR kinase inhibitor (TOR-KI) MLN0128 with SBI-756, a compound targeting eukaryotic translation initiation factor 4G1 (eIF4G1), a scaffolding protein in the eIF4F complex. Results Treatment of DLBCL and MCL cells with SBI-756 synergised with venetoclax to induce apoptosis in vitro, and enhanced venetoclax efficacy in vivo. SBI-756 prevented eIF4E-eIF4G1 association and cap-dependent translation without affecting mTOR substrate phosphorylation. In TOR-KI-resistant DLBCL cells lacking eIF4E binding protein-1, SBI-756 still sensitised to venetoclax. SBI-756 selectively reduced translation of mRNAs encoding ribosomal proteins and translation factors, leading to a reduction in protein synthesis rates in sensitive cells. When normal lymphocytes were treated with SBI-756, only B cells had reduced viability, and this correlated with reduced protein synthesis. Conclusions Our data highlight a novel combination for treatment of aggressive lymphomas, and establishes its efficacy and selectivity using preclinical models.