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

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

Selection of start codon during mRNA scanning in eukaryotic translation initiation.

Accurate and high-speed scanning and subsequent selection of the correct start codon are important events in protein synthesis. Eukaryotic mRNAs have long 5' UTRs that are inspected for the presence of a start codon by the ribosomal 48S pre-initiation complex (PIC). However, the conformational state of the 48S PIC required for inspecting every codon is not clearly understood. Here, atomistic molecular dynamics (MD) simulations and energy calculations suggest that the scanning conformation of 48S PIC may reject all but 4 (GUG, CUG, UUG and ACG) of the 63 non-AUG codons, and initiation factor eIF1 is crucial for this discrimination. We provide insights into the possible role of initiation factors eIF1, eIF1A, eIF2α and eIF2ß in scanning. Overall, the study highlights how the scanning conformation of ribosomal 48S PIC acts as a coarse selectivity checkpoint for start codon selection and scans long 5' UTRs in eukaryotic mRNAs with accuracy and high speed.

eIF5B and eIF1A reorient initiator tRNA to allow ribosomal subunit joining.

Translation initiation defines the identity and quantity of a synthesized protein. The process is dysregulated in many human diseases1,2. A key commitment step is when the ribosomal subunits join at a translation start site on a messenger RNA to form a functional ribosome. Here, we combined single-molecule spectroscopy and structural methods using an in vitro reconstituted system to examine how the human ribosomal subunits join. Single-molecule fluorescence revealed when the universally conserved eukaryotic initiation factors eIF1A and eIF5B associate with and depart from initiation complexes. Guided by single-molecule dynamics, we visualized initiation complexes that contained both eIF1A and eIF5B using single-particle cryo-electron microscopy. The resulting structure revealed how eukaryote-specific contacts between the two proteins remodel the initiation complex to orient the initiator aminoacyl-tRNA in a conformation compatible with ribosomal subunit joining. Collectively, our findings provide a quantitative and architectural framework for the molecular choreography orchestrated by eIF1A and eIF5B during translation initiation in humans.

Conformational rearrangements upon start codon recognition in human 48S translation initiation complex.

Selection of the translation start codon is a key step during protein synthesis in human cells. We obtained cryo-EM structures of human 48S initiation complexes and characterized the intermediates of codon recognition by kinetic methods using eIF1A as a reporter. Both approaches capture two distinct ribosome populations formed on an mRNA with a cognate AUG codon in the presence of eIF1, eIF1A, eIF2-GTP-Met-tRNAiMet and eIF3. The 'open' 40S subunit conformation differs from the human 48S scanning complex and represents an intermediate preceding the codon recognition step. The 'closed' form is similar to reported structures of complexes from yeast and mammals formed upon codon recognition, except for the orientation of eIF1A, which is unique in our structure. Kinetic experiments show how various initiation factors mediate the population distribution of open and closed conformations until 60S subunit docking. Our results provide insights into the timing and structure of human translation initiation intermediates and suggest the differences in the mechanisms of start codon selection between mammals and yeast.

Lnc5926 is essential for early embryonic development in goats through regulation of ZSCAN4 and EIF1AX.

Long noncoding RNAs (lncRNAs) are abundant in mammalian genomes and have been found to play important roles in many biological events. However, the mechanism by which lncRNAs regulate embryonic development remains to be fully elucidated. Here, we investigated the function of the lncRNA, TCONS_00135926 (referred to as lnc5926), through knockdown and overexpression experiments in goat early embryos. Lnc5926 expression at the eight-cell embryonic stage was significantly higher than that at other stages, which was consistent with the pattern of embryonic genome activation (EGA) gene expression. The blastocyst rate after lnc5926 knockdown in eight-cell embryos was significantly lower than that in the control group (0.2% vs. 17.1%, p < 0.05), whereas the cleavage rate was not affected (71.9% vs. 75.1%, p ˃ 0.05). After knockdown or overexpression of lnc5926 in embryos, we measured expression levels of the potential target genes, STAM, HACD1, UBL5, MIOX, ELF1, and the key EGA genes, ZSCAN4 and EIF1AX. Only ZSCAN4 and EIF1AX were significantly downregulated after lnc5926 knockdown, and this effect was reversed by lnc5926 overexpression. We conclude that lnc5926 plays an essential role in early embryonic development in goats by regulating expression of EGA-associated genes.

XPO1-Mediated EIF1AX Cytoplasmic Relocation Promotes Tumor Migration and Invasion in Endometrial Carcinoma.

Dysregulation of eukaryotic translation initiation factor 1A, X-linked (EIF1AX), has been implicated in the pathogenesis of some cancers. However, the role of EIF1AX in endometrial carcinoma (EC) remains unknown. We investigated the EIF1AX expression in EC patients and assessed its tumorigenesis-associated function and nucleocytoplasmic transport mechanism in vitro and in vivo. The results indicated that the cytoplasmic EIF1AX expression showed a gradual increase when going from endometrium normal tissue, simple endometrial hyperplasia, complex endometrial hyperplasia, and endometrial atypical hyperplasia to EC, while vice versa for the nuclear EIF1AX expression. In addition, the cytoplasmic EIF1AX expression was positively correlated with histologic type, high International Federation of Gynecology and Obstetrics (FIGO) grade, advanced FIGO stage, deeper infiltration, high Ki67 index, and shorter recurrence-free survival in EC patients. In vitro, short hairpin RNA-mediated EIF1AX depletion or SV40NLS-mediated EIF1AX import into the nucleus in multiple human EC cells potently suppressed cell migration and invasion, epithelial-mesenchymal transition, and lung metastasis. Moreover, exportin 1 induced the transport of EIF1AX from the nucleus to the cytoplasm that could be inhibited by leptomycin B treatment or the mutation in the EIF1AX location sequence. These results demonstrate that cytoplasmic EIF1AX may play a key role in the incidence and promotion of EC, and thus, targeting EIF1AX or its nucleocytoplasmic transport process may offer an effective new therapeutic approach to EC.

Magnaporthe oryzae as an expression host for the production of the unspecific peroxygenase AaeUPO from the basidiomycete Agrocybe aegerita.

The filamentous fungus Magnaporthe oryzae has the potential to be developed as an alternative platform organism for the heterologous production of industrially important enzymes. M. oryzae is easy to handle, fast-growing and unlike yeast, posttranslational modifications like N-glycosylations are similar to the human organism. Here, we established M. oryzae as a host for the expression of the unspecific peroxygenase from the basidiomycete Agrocybe aegerita (AaeUPO). Note, UPOs are attractive biocatalysts for selective oxyfunctionalization of non-activated carbon-hydrogen bonds. To improve and simplify the isolation of AaeUPO in M. oryzae, we fused a Magnaporthe signal peptide for protein secretion and set it under control of the strong EF1α-promoter. The success of the heterologous production of full-length AaeUPO in M. oryzae and the secretion of the functional enzyme was confirmed by a peroxygenase-specific enzyme assay. These results offer the possibility to establish the filamentous ascomycete M. oryzae as a broad applicable alternative expression system.

Nuclear-localized eukaryotic translation initiation factor 1A is involved in mouse preimplantation embryo development.

Preimplantation embryo development is characterized by drastic nuclear reprogramming and dynamic stage-specific gene expression. Key regulators of this earliest developmental stage have not been revealed. In the present study, a "non-classical" nuclear-localization pattern of eIF1A was observed during early developmental stages of mouse preimplantation embryo before late-morula. In particular, eIF1A is most highly expressed in the nuclear of 2-cell embryo. Knockdown eIF1A by siRNA microinjection affected the development of mouse preimplantation embryo, resulted in decreased blastocyst formation rate. CDX2 protein expression level significantly down-regulated after eIF1A knockdown in morula stage. In addition, the mRNA expression level of Hsp70.1 was also decreased in 2-cell embryo. The results indicate an indispensable role of eIF1A in mouse preimplantation embryos.

Bioinformatics analysis of GNAQ, GNA11, BAP1, SF3B1,SRSF2, EIF1AX, PLCB4, and CYSLTR2 genes and their role in the pathogenesis of Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, and its metastases are known to be fatal. It is critical to identify molecular markers to be used in potential prognostic evaluation for early diagnosis, treatment, and metastasis or to investigate all aspects of known genetic anomalies. Therefore, this study aimed to analyze the eight genes (GNAQ, GNA11, BAP1, SF3B1, SRSF2, EIF1AX, PLCB4, and CYSLTR2) that are associated with the most common genetic anomalies in UM from a molecular perspective. The genome sequences and expression profiles of 108 UM patients were obtained via bioinformatics tools that provide data from TCGA. The overall mutational load and the mutation patterns for eight genes, in particular, were thoroughly determined. Moreover, PolyPhen2 and SNAP2 tools were used to estimate the oncogenic/pathogenic properties of identified mutations for UM. In addition to the mutation profile, the effects of the presence of a mutation on gene expression and survival were determined. Finally, STRING network analysis was performed to better understand the functional relationships of mutated proteins in cellular processes. There were 27 missense mutations, 16 frameshift mutations, six nonsense mutations, and three splice region mutations among the 52 mutations found in eight genes, and 26 of them had pathogenic properties. BAP1 m-RNA expression was significantly lower in tumors with the mutant genotype (p = .001). The impact of gene expression, which has poor prognostic importance, on survival is statistically significant for high-expressed BAP1 (p = .0015) and low-expressed CYSLTR2 (p = .0021). To assess the current state of this potentially devastating disease, a molecular perspective has been evaluated. Defining this molecular perspective can be useful in developing targeted drug therapies and personalized medicine.

Characterization and Clinical Significance of EIF1AX Mutations and Co-Mutations in Cytologically Indeterminate Thyroid Nodules: A 5-Year Retrospective Analysis.

OBJECTIVE: Mutations in the EIF1AX gene have been recently detected in a small percentage of benign and malignant thyroid lesions. We sought to investigate the prevalence and clinical significance of EIF1AX mutations and co-mutations in cytologically indeterminate thyroid nodules at our institution. MATERIALS AND METHODS: A 5-year retrospective analysis was performed on thyroid nodules with a cytologic diagnosis of Bethesda category III or IV, which had undergone testing by our in-house next generation sequencing panel. Surgically resected nodules with EIF1AX mutations were identified, and mutation type and presence of co-mutations were correlated with histopathologic diagnosis. RESULTS: 41/904 (4.5%) cases overall and 26/229 (11.4%) surgically resected nodules harbored an EIF1AX mutation. The most common histologic diagnoses were follicular thyroid carcinoma and follicular variant of papillary thyroid carcinoma. 11/26 (42.3%) of nodules had isolated EIF1AX mutation. Comutations were found in RAS (12/26; 46.2%), TERT (5/26; 19.2%) and TP53 (2/26; 7.7%). EIF1AX mutation alone conferred a 36.4% risk of malignancy (ROM) and 54.5% ROM or noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), while the ROM was significantly higher in nodules with concurrent RAS (71.4%), TERT, TP53 and RAS+TERT (100%) mutations. CONCLUSION: EIF1AX mutations occur in benign and malignant follicular thyroid neoplasms. In our cohort, the majority of mutations occurred at the splice acceptor site between exons 5 and 6. Importantly, the coexistence of EIF1AX mutations with other driver pathogenic mutations in RAS, TERT and TP53 conferred a 100% ROM or NIFTP, indicating that such nodules require surgical removal.

The Genomic Landscape of Thyroid Cancer Tumourigenesis and Implications for Immunotherapy.

Thyroid cancer is the most prevalent endocrine malignancy that comprises mostly indolent differentiated cancers (DTCs) and less frequently aggressive poorly differentiated (PDTC) or anaplastic cancers (ATCs) with high mortality. Utilisation of next-generation sequencing (NGS) and advanced sequencing data analysis can aid in understanding the multi-step progression model in the development of thyroid cancers and their metastatic potential at a molecular level, promoting a targeted approach to further research and development of targeted treatment options including immunotherapy, especially for the aggressive variants. Tumour initiation and progression in thyroid cancer occurs through constitutional activation of the mitogen-activated protein kinase (MAPK) pathway through mutations in BRAF, RAS, mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathway and/or receptor tyrosine kinase fusions/translocations, and other genetic aberrations acquired in a stepwise manner. This review provides a summary of the recent genetic aberrations implicated in the development and progression of thyroid cancer and implications for immunotherapy.

Iron overload induces apoptosis of osteoblast cells via eliciting ER stress-mediated mitochondrial dysfunction and p-eIF2α/ATF4/CHOP pathway in vitro.

Iron is an essential element for crucial biological function; whereas excess iron sedimentation impairs the main functions of tissues or organs. Cumulative researches have shown that the disturbances in iron metabolism, especially iron overload is closely concatenating with bone loss. Nevertheless, the specific process of iron overload-induced apoptosis in osteoblasts has not been thoroughly studied. In this study, our purpose is to elucidate the mechanism of osteoblast apoptosis induced by iron overload via the MC3T3-E1 cell line. Ferric ammonium citrate (FAC) was utilized to simulate iron overload conditions in vitro. These results showed that treatment with FAC dose-dependently induced the apoptosis of MC3T3-E1 cells at 48 h, dysfunction of iron metabolism, and increased intracellular reactive oxygen species (ROS) levels. Following, FAC does-dependently caused the calcium dyshomeostasis, decreased the calcium concentration in endoplasmic reticulum (ER), but increased the crosstalk between ER and mitochondria, and calcium concentration in the mitochondria. Moreover, FAC dose-dependently decreased mitochondrial membrane potential (MMP) and enhanced the expression of apoptosis related proteins (Bax, Cyto-C and C-caspase3). We furthermore revealed that FAC treatment activated the ER-mediated cell apoptosis via p-eIF2α/ATF4/CHOP pathway in MC3T3-E1 osteoblasts cells. In addition, pretreatment with the N-acetylcysteine (NAC) or Tauroursodeoxycholate Sodium (TUDC) attenuated cell apoptosis, ROS levels, mitochondria fragmentation and ER stress-related protein expression, and recovered the protein expression related to iron metabolism. In conclusion, our finding suggested that iron overload induced apoptosis via eliciting ER stress, which resulted in mitochondrial dysfunction and activated p-eIF2α/ATF4/CHOP pathway.

Candidate reference genes for quantitative gene expression analysis in Lagerstroemia indica.

Quantitative gene expression analysis by qPCR requires reference genes for normalization. Lagerstroemia indica (crape myrtle) is a popular ornamental plant in the world, but suitable endogenous reference genes are lacking. To find suitable reference genes, we evaluated the stabilities of nine candidate genes in six experimental data sets: six different tissues, three leaf colors, nine flower colors, and under three abiotic stresses (salt, drought, cold) using four statistical algorithms. A target gene LiMYB56 (homolog of Arabidopsis MYB56) was used to verify the authenticity and accuracy of the candidate reference genes. The results showed that the combination of two stably expressed reference genes, rather than a single reference gene, improved the accuracy of the qPCR. LiEF1α-2 + LiEF1α-3 was best for the tissue, salt treatment, and drought treatment sets; LiEF1α-2 + LiEF1α-1 was optimal for leaf color; LiEF1α-2 + LiACT7 was optimal for cold treatment; and LiUBC + LiEF1α-1 was best for the flower color set. Notably, LiEF1α-2 had high expression stability in all six experimental sets, implying it may be a good reference gene for expression studies in L. indica. Our results will facilitate future gene expression studies in L. indica.

Transcriptional repression of p21 by EIF1AX promotes the proliferation of breast cancer cells.

OBJECTIVE: Dysregulation of the cell cycle is associated with the progression of malignant cancer, but its precise functional contribution is unknown. MATERIALS AND METHODS: The expression of EIF1AX in breast cancer tissues was detected by qRT-PCR and immunohistochemistry staining. Colony formation and tumour xenograft assays were used to examine the tumorigenesis-associated function of EIF1AX in vitro and in vivo. RNA-Seq analysis was used to select the downstream target genes of EIF1AX. Flow cytometry, ChIP and luciferase assays were used to investigate the molecular mechanisms by which EIF1AX regulates p21 in breast cancer cells. RESULTS: EIF1AX promoted breast cancer cell proliferation by promoting the G1/S cell cycle transition. A mechanistic investigation showed that EIF1AX inhibited the expression of p21, which is an essential cell cycle regulator. We identified that the transcriptional regulation of p21 by EIF1AX was p53-independent. Clinically, EIF1AX levels were significantly elevated in breast cancer tissues, and the high level of EIF1AX was associated with lower survival rates in breast cancer patients. CONCLUSIONS: Our results imply that EIF1AX may play a key role in the incidence and promotion of breast cancer and may, thus, serve as a valuable target for breast cancer therapy.

Somatic Mutations in the BRAF, KRAS, NRAS, EIF1AX, and TERT Genes: Diagnostic Value in Thyroid Neoplasms.

The feasibility of using molecular genetic markers associated with thyroid neoplasms and more aggressive course of the disease is now actively studied. We analyzed the diagnostic value of somatic mutations in the hot spots of BRAF, KRAS, KRAS, EIF1AX, and TERT genes in histological material from 153 patients with thyroid gland neoplasms. BRAF mutations (exon 15, codon area 600-601) were found in 54 patients, NRAS mutations (exon 3, codon 61) were detected in 12 patients; mutations KRAS, TERT, and EIF1AX genes were not detected.

Lack of methylation on transgene leads to high level and persistent transgene expression in induced pluripotent stem cells.

Short-lived therapeutic gene expression in mammalian cells by DNA methylation is one of the major challenges in gene therapy. In this study, we assessed the implication of DNA methylation on the duration of GFP expression in mouse embryonic stem (ES) and mouse induced pluripotent stem (iPS) cells. The cells were transduced with lentivirus (LV) carrying green fluorescent protein (GFP) driven by either human elongation factor (EF1α) or cytomegalovirus (CMV) promoter. Transduced iPS cells exhibited higher percentage of GFP+ cells with persistent mean fluorescent intensity than transduced ES cells. Analysis on the integrated copy of transgene in the population of the transduced cells demonstrated similar copy number. However, significant increase in GFP intensity following 5-azaC treatment was observed in transduced ES cells only, suggesting the influence of DNA methylation in transgene silencing. Subsequent DNA methylation analysis showed that the promoter and the GFP region of the provirus in iPS cells had negligible methylation profile compared to transduced ES cells. Interestingly, sustained transgene expression was observed upon directed differentiation of transduced iPS cells towards CD34+ CD45+ cells. Hence, this study has shown that favourable transgene activity from lentiviral transduced iPS cells was due to the lack of methylation at the proviral regions.

Determination of reference genes for ovine pulmonary adenocarcinoma infected lung tissues using RNA-seq transcriptome profiling.

Ovine pulmonary adenocarcinoma (OPA) is a globally occurring tumor of lung epithelium which seriously affects the development of sheep farming. In our research, lung tissues of 3 naturally infected OPA individuals and 3 healthy individuals (2-4 years old) were collected. RNA was extracted for transcriptome analysis and reference gene selection. According to transcriptome analysis, 7 candidate reference genes (eukaryotic translation initiation factor 1, EIF1; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; beta-actin, ACTB; GABA Type A receptor-associated protein, GABARAP; activating transcription factor 4, ATF4; ribosomal protein S15, RPS15; and Y-Box binding protein 1, YBX1) showed fragments per kilobase of transcript per million fragments mapped (FPKM) values > 200.0 and standard errors of the means (SEM) < 20.0. Expression of the above candidate reference genes was evaluated by Real-time quantitative polymerase chain reaction (RT-qPCR) combined with the analysis using GeNorm, NormFinder, and BestKeeper software. Comprehensive analysis of the results showed that ACTB was the most stable one, followed by EIF1 and GABARAP. Then, expression stability of the above three genes were validated, suggesting as suitable reference genes in sheep lung tissue, in additional 30 OPA-affected lung tissues and 10 healthy ovine lung tissues. Finally, our findings will be helpful for the subsequent study on the tumorigenic mechanism of OPA.

Structural Insights into the Mammalian Late-Stage Initiation Complexes.

In higher eukaryotes, the mRNA sequence in the direct vicinity of the start codon, called the Kozak sequence (CRCCaugG, where R is a purine), is known to influence the rate of the initiation process. However, the molecular basis underlying its role remains poorly understood. Here, we present the cryoelectron microscopy (cryo-EM) structures of mammalian late-stage 48S initiation complexes (LS48S ICs) in the presence of two different native mRNA sequences, ß-globin and histone 4, at overall resolution of 3 and 3.5 Å, respectively. Our high-resolution structures unravel key interactions from the mRNA to eukaryotic initiation factors (eIFs): 1A, 2, 3, 18S rRNA, and several 40S ribosomal proteins. In addition, we are able to study the structural role of ABCE1 in the formation of native 48S ICs. Our results reveal a comprehensive map of ribosome/eIF-mRNA and ribosome/eIF-tRNA interactions and suggest the impact of mRNA sequence on the structure of the LS48S IC.

EIF1AX c.338-2A>T splice site mutation in a patient with trabecular adenoma and cytological indeterminate lesion.

The EIF1AX gene mutations have been recently associated with papillary thyroid carcinoma and anaplastic thyroid cancer. According with these reports, the gene as been considered as a drive gene for thyroid cancer development. However, the occurrence of these alterations in benign thyroid lesions is not known and is still under investigation. Some authors have already reported the presence of EIF1AX variants in follicular adenomas and hyperplastic nodules. Here, we describe for the first time a case of a man with the EIF1AX c.338-2A>T splice site mutation in an indeterminate FNA lesion with trabecular adenoma at final histology in the absence of other pathogenetic mutations, demonstrating that further studies are required to better understand EIF1AX role in the tumorigenesis of thyroid carcinoma.

EIF1AX c.338-2A>T splice site mutation in a patient with trabecular adenoma and cytological indeterminate lesion

SUMMARY The EIF1AX gene mutations have been recently associated with papillary thyroid carcinoma and anaplastic thyroid cancer. According with these reports, the gene as been considered as a drive gene for thyroid cancer development. However, the occurrence of these alterations in benign thyroid lesions is not known and is still under investigation. Some authors have already reported the presence of EIF1AX variants in follicular adenomas and hyperplastic nodules. Here, we describe for the first time a case of a man with the EIF1AX c.338-2A>T splice site mutation in an indeterminate FNA lesion with trabecular adenoma at final histology in the absence of other pathogenetic mutations, demonstrating that further studies are required to better understand EIF1AX role in the tumorigenesis of thyroid carcinoma.