Translational Apparatus Research Articles

Trinity of G-tetrads and origin of translation

BackgroundThe RNA world hypothesis cannot address most of the questions of the origin of life without violating the continuity principle (small Darwinian steps without foresight and miracles). Moreover, the RNA world is an isolated system incapable of accommodating the genetic code and evolving into extant biochemistry. All these problems are rooted in the central assumption of the hypothesis: de novo appearance of the ribozymes, production of which represents a multistep reaction requiring the complementarity principle. Thus, even the basis of the RNA world is at odds with the continuity principle—it uses foresight (multistep reaction) and a miracle (complementarity principle). Can a three-dimensional (3D) architecture, capable of molecular recognition and catalysis, be formed in a single-step reaction without the complementarity or any other preexisting rules?HypothesisAt first glance, the above question sounds rhetoric since the complementarity principle is the essential feature of the RNA world; it turns an RNA polymer into a genetic material. Without it, the RNA world becomes as shapeless and unconvincing as other hypotheses based on the non-hereditary molecules (i.e., protein world). However, it was suggested recently that the quadruplexes could initiate life and take necessary evolutionary steps before the arrival of the complementarity rules. The hypothesis relies on the unique properties of guanines (Gs) to self-assemble into G-tetrads and efficiently polymerize without any external help or preexisting rules. Interestingly, polyG folds into an unusually stable and well-structured monomolecular architecture that uses the quadruplex domain (QD) assembly. The QD has a strictly defined zigzag-like building pattern to accommodate only three G-tetrads. Since both QD architecture and codon length are based on triplets, the inevitable question arises: are they related? Or could QD play the role of the early adapter and determine the codon length? The current paper is an attempt to answer this question.ConclusionWhile without translation apparatus most of the steps of the extant translation are physically impossible, the QD-mediated translation is sterically feasible and can be explained by physicochemical properties of the QD and the amino acids without violating the continuity principle. Astonishingly, the quadruplex world hypothesis can address all the shortcomings of the RNA world, including its most significant challenge—step-by-step evolution from the polymerization of the first polynucleotide to the extant biochemistry.

Mitochondrial rRNA Methylation by Mettl15 Contributes to the Exercise and Learning Capability in Mice.

Mitochondrial translation is a unique relic of the symbiotic origin of the organelle. Alterations of its components cause a number of severe human diseases. Hereby we report a study of mice devoid of Mettl15 mitochondrial 12S rRNA methyltransferase, responsible for the formation of m4C839 residue (human numbering). Homozygous Mettl15−/− mice appeared to be viable in contrast to other mitochondrial rRNA methyltransferase knockouts reported earlier. The phenotype of Mettl15−/− mice is much milder than that of other mutants of mitochondrial translation apparatus. In agreement with the results obtained earlier for cell cultures with an inactivated Mettl15 gene, we observed accumulation of the RbfA factor, normally associated with the precursor of the 28S subunit, in the 55S mitochondrial ribosome fraction of knockout mice. A lack of Mettl15 leads to a lower blood glucose level after physical exercise relative to that of the wild-type mice. Mettl15−/− mice demonstrated suboptimal muscle performance and lower levels of Cox3 protein synthesized by mitoribosomes in the oxidative soleus muscles. Additionally, we detected decreased learning capabilities in the Mettl15−/− knockout mice in the tests with both positive and negative reinforcement. Such properties make Mettl15−/− knockout mice a suitable model for mild mitochondriopathies.

HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins

The biological role of RNA-binding proteins in the secretory pathway is not well established. Here, we describe that human HDLBP/Vigilin directly interacts with more than 80% of ER-localized mRNAs. PAR-CLIP analysis reveals that these transcripts represent high affinity HDLBP substrates and are specifically bound in their coding sequences (CDS), in contrast to CDS/3’UTR-bound cytosolic mRNAs. HDLBP crosslinks strongly to long CU-rich motifs, which frequently reside in CDS of ER-localized mRNAs and result in high affinity multivalent interactions. In addition to HDLBP-ncRNA interactome, quantification of HDLBP-proximal proteome confirms association with components of the translational apparatus and the signal recognition particle. Absence of HDLBP results in decreased translation efficiency of HDLBP target mRNAs, impaired protein synthesis and secretion in model cell lines, as well as decreased tumor growth in a lung cancer mouse model. These results highlight a general function for HDLBP in the translation of ER-localized mRNAs and its relevance for tumor progression.

The natural bicyclic hexapeptide RA-VII is a novel inhibitor of the eukaryotic translocase eEF2

A cyclic hexapeptide, RA-VII isolated from the Rubiaceae family of plants, has high cytotoxic activity. Although RA-VII has been shown to inhibit protein synthesis in eukaryotic cells, the molecular mode of its action is not clear. Here we investigate the mechanism of the RAVII action on the translation apparatus. Biochemical functional assays showed that RA-VII inhibits poly(U)-dependent polyphenylalanine synthesis in the presence of animal elongation factors eEF1A and eEF2. Furthermore, RAVII prevented eEF2/ribosome-dependent GTPase activity, but not eEF-1A/ribosome-dependent activity. A filter binding assay demonstrated that RA-VII markedly enhances the binding affinity of eEF2 for GTP, but not for GDP, and prevents exchange of GTP in the eEF2-GTP complex, even after addition of a large excess of GTP/GDP. Limited proteolysis experiments indicated that RA-VII prevents the digestion of eEF2 in the presence of either GTP or GMPPCP, but not with GDP. Further footprint analysis and a translocation assay showed that the eEF2•GMPPNP•RA-VII complex binds to the conserved rRNA regions at the factor-binding center of the ribosome and retains the ability to translocate the A site-bound tRNA to the P-site. These results suggest that RA-VII tightly stabilizes the GTP•eEF2 complex structure, which is able to bind to the ribosomal functional site, but seems to suppress normal turnover of eEF2 after translocation. The properties of RA-VII make it a novel ligand for probing the action of eEF2 in the process of translocation on the ribosome.

Hyperaccurate Ribosomes for Improved Genetic Code Reprogramming.

The reprogramming of the genetic code through the introduction of noncanonical amino acids (ncAAs) has enabled exciting advances in synthetic biology and peptide drug discovery. Ribosomes that function with high efficiency and fidelity are necessary for all of these efforts, but for challenging ncAAs, the competing processes of near-cognate readthrough and peptidyl-tRNA dropoff can be issues. Here we uncover the surprising extent of these competing pathways in the PURE translation system using mRNAs encoding peptides with affinity tags at the N- and C-termini. We also show that hyperaccurate or error restrictive ribosomes with mutations in ribosomal protein S12 lead to significant improvements in yield and fidelity in the context of both canonical AAs and a challenging α,α-disubstituted ncAA. Hyperaccurate ribosomes also improve yields for quadruplet codon readthrough for a tRNA containing an expanded anticodon stem-loop, although they are not able to eliminate triplet codon reading by this tRNA. The impressive improvements in fidelity and the simplicity of introducing this mutation alongside other efforts to engineer the translation apparatus make hyperaccurate ribosomes an important advance for synthetic biology.

Ribosomal protein S18 acetyltransferase RimI is responsible for the acetylation of elongation factor Tu

N-terminal acetylation is widespread in the eukaryotic proteome but in bacteria is restricted to a small number of proteins mainly involved in translation. It was long known that elongation factor Tu (EF-Tu) is N-terminally acetylated, whereas the enzyme responsible for this process was unclear. Here, we report that RimI acetyltransferase, known to modify ribosomal protein S18, is likewise responsible for N-acetylation of the EF-Tu. With the help of inducible tufA expression plasmid, we demonstrated that the acetylation does not alter the stability of EF-Tu. Binding of aminoacyl tRNA to the recombinant EF-Tu in vitro was found to be unaffected by the acetylation. At the same time, with the help of fast kinetics methods, we demonstrate that an acetylated variant of EF-Tu more efficiently accelerates A-site occupation by aminoacyl-tRNA, thus increasing the efficiency of in vitro translation. Finally, we show that a strain devoid of RimI has a reduced growth rate, expanded to an evolutionary timescale, and might potentially promote conservation of the acetylation mechanism of S18 and EF-Tu. This study increased our understanding of the modification of bacterial translation apparatus.

Adaptation of the protein translational apparatus during ATDC5 chondrogenic differentiation.

IntroductionRibosome biogenesis is integrated with many cellular processes including proliferation, differentiation and oncogenic events. Chondrogenic proliferation and differentiation require a high cellular translational capacity to facilitate cartilaginous extracellular matrix production. We here investigated the expression dynamics of factors involved in ribosome biogenesis during in vitro chondrogenic differentiation and determined whether protein translation capacity adapts to different phases of chondrogenic differentiation.MaterialsSnoRNA expression during ATDC5 differentiation was analyzed by RNA sequencing of samples acquired from day 0 (progenitor stage), 7 (chondrogenic stage) and day 14 (hypertrophic stage). RT-qPCR was used to determine expression of fibrillarin, dyskerin, UBF-1, Sox9, Col2a1, Runx2, Col10a1 mRNAs and 18S, 5.8S and 28S rRNAs. Protein expression of fibrillarin, dyskerin and UBF-1 was determined by immunoblotting. Ribosomal RNA content per cell was determined by calculating rRNA RT-qPCR signals relative to DNA content (SYBR Green assay). Total protein translational activity was evaluated with a puromycilation assay and polysome profiling.ResultsAs a result of initiation of chondrogenic differentiation (Δt0-t7), 21 snoRNAs were differentially expressed (DE). Hypertrophic differentiation caused DE of 23 snoRNAs (Δt7-t14) and 43 when t0 was compared to t14. DE snoRNAs, amongst others, target nucleotide modifications in the 28S rRNA peptidyl transferase center and the 18S rRNA decoding center. UBF-1, fibrillarin and dyskerin expression increased as function of differentiation and displayed highest fold induction at day 5–6 in differentiation. Ribosomal RNA content per cell was significantly increased at day 7, but not at day 14 in differentiation. Similar dynamics in translational capacity and monosomal ribosome fraction were observed during differentiation.ConclusionThe expression of a great number of ribosome biogenesis factors is altered during chondrogenic differentiation of ATDC5 cells, which is accompanied by significant changes in cellular translational activity. This elucidation of ribosome biogenesis dynamics in chondrogenic differentiation models enables the further understanding of the role of ribosome biogenesis and activity during chondrocyte cell commitment and their roles in human skeletal development diseases.

Significant impact of mTORC1 and ATF4 pathways in CHO cell recombinant protein production induced by CDK4/6 inhibitor.

The CDK4/6 inhibitor has been shown to increase recombinant protein productivity in Chinese hamster ovary (CHO) cells. Therefore, we investigated the mechanism that couples cell-cycle inhibitor (CCI) treatment with protein productivity utilizing proteomics and phosphoproteomics. We identified mTORC1 as a critical early signaling event that preceded boosted productivity. Following CCI treatment, mTOR exhibited a transient increase in phosphorylation at a novel site that is also conserved in humans and mouse. Upstream of mTORC1, increased phosphorylation of AKT1S1 and decreased phosphorylation of RB1 may provide molecular links between CDK4/6 inhibition and mTORC1. Downstream, increased EIF4EBP1 phosphorylation was observed, which can mediate cap-dependent translation. In addition, the collective effect of increased phosphorylation of RPS6, increased phosphorylation of regulators of RNA polymerase I, and increased protein expression in the transfer RNA-aminoacylation pathway may contribute to enhancing the translational apparatus for increased productivity. In concert, an elevated stress response via GCN2/EIF2AK4-ATF4 axis persisted over the treatment course, which may link mTOR to downstream responses including the unfolded protein responseand autophagy to enhance proper protein folding and secretion. Together, this comprehensive proteomics and phosphoproteomics characterization of CCI-treated CHO cells offers insights into understanding multiple aspects of signaling events resulting from CDK4/CDK6 inhibition.

Detection methods targeting the positive- and negative-sense RNA transcripts from plus-stranded RNA viruses.

The largest group of viruses in the Baltimore classification system comprises viruses with a positive‐sense, single‐stranded RNA genome. Once the viral genome is released into the cytoplasm of a specific host cell following virus entry, it functions directly as an mRNA, and the virus‐encoded proteins that are essential for genome replication are produced by the translation apparatus of the host cell. The positive‐sense genome is replicated in two stages, initially the positive strand is copied to make a negative‐sense RNA, which then functions as the template for transcription of many new positive‐sense genomes. Virus infections can be detected at different stages throughout the infection cycle for diagnostic and scientific purposes. Here, the advantages and disadvantages of some of the relevant methods for genome detection will be briefly reviewed with special emphasis on techniques allowing strand‐specific RNA detection. Furthermore, tools of the future are considered.

Eukaryotic ribosome quality control system: a potential therapeutic target for human diseases.

Protein homeostasis is well accepted as the prerequisite for proper operation of various life activities. As the main apparatus of protein translation, ribosomes play an indispensable role in the maintenance of protein homeostasis. Nevertheless, upon stimulation of various internal and external factors, malfunction of ribosomes may be evident with the excessive production of aberrant proteins, accumulation of which can result in deleterious effects on cellular fate and even cell death. Ribosomopathies are characterized as a series of diseases caused by abnormalities of ribosomal compositions and functions. Correspondingly, cell evolves several ribosome quality control mechanisms in maintaining the quantity and quality of intracellular ribosomes, namely ribosome quality control system (RQCS). Of note, RQCS can tightly monitor the entire process from ribosome biogenesis to its degradation, with the capacity of coping with ribosomal dysfunction, including misassembled ribosomes and incorrectly synthesized ribosomal proteins. In the current literature review, we mainly introduce the RQCS and elaborate on the underlying pathogenesis of several ribosomopathies. With the in-depth understanding of ribosomal dysfunction and molecular basis of RQCS, therapeutic strategy by specifically targeting RQCS remains a promising option in treating patients with ribosomopathies and other ribosome-associated human diseases.

Homeostatic scaling is driven by a translation-dependent degradation axis that recruits miRISC remodeling.

Homeostatic scaling in neurons has been attributed to the individual contribution of either translation or degradation; however, there remains limited insight toward understanding how the interplay between the two processes effectuates synaptic homeostasis. Here, we report that a codependence between protein synthesis and degradation mechanisms drives synaptic homeostasis, whereas abrogation of either prevents it. Coordination between the two processes is achieved through the formation of a tripartite complex between translation regulators, the 26S proteasome, and the miRNA-induced silencing complex (miRISC) components such as Argonaute, MOV10, and Trim32 on actively translating transcripts or polysomes. The components of this ternary complex directly interact with each other in an RNA-dependent manner. Disruption of polysomes abolishes this ternary interaction, suggesting that translating RNAs facilitate the combinatorial action of the proteasome and the translational apparatus. We identify that synaptic downscaling involves miRISC remodeling, which entails the mTORC1-dependent translation of Trim32, an E3 ligase, and the subsequent degradation of its target, MOV10 via the phosphorylation of p70 S6 kinase. We find that the E3 ligase Trim32 specifically polyubiquitinates MOV10 for its degradation during synaptic downscaling. MOV10 degradation alone is sufficient to invoke downscaling by enhancing Arc translation through its 3' UTR and causing the subsequent removal of postsynaptic AMPA receptors. Synaptic scaling was occluded when we depleted Trim32 and overexpressed MOV10 in neurons, suggesting that the Trim32-MOV10 axis is necessary for synaptic downscaling. We propose a mechanism that exploits a translation-driven protein degradation paradigm to invoke miRISC remodeling and induce homeostatic scaling during chronic network activity.

Localization and Functional Roles of Components of the Translation Apparatus in the Eukaryotic Cell Nucleus.

Components of the translation apparatus, including ribosomal proteins, have been found in cell nuclei in various organisms. Components of the translation apparatus are involved in various nuclear processes, particularly those associated with genome integrity control and the nuclear stages of gene expression, such as transcription, mRNA processing, and mRNA export. Components of the translation apparatus control intranuclear trafficking; the nuclear import and export of RNA and proteins; and regulate the activity, stability, and functional recruitment of nuclear proteins. The nuclear translocation of these components is often involved in the cell response to stimulation and stress, in addition to playing critical roles in oncogenesis and viral infection. Many components of the translation apparatus are moonlighting proteins, involved in integral cell stress response and coupling of gene expression subprocesses. Thus, this phenomenon represents a significant interest for both basic and applied molecular biology. Here, we provide an overview of the current data regarding the molecular functions of translation factors and ribosomal proteins in the cell nucleus.

LIN28B alters ribosomal dynamics to promote metastasis in MYCN-driven malignancy.

High expression of LIN28B is associated with aggressive malignancy and poor survival. Here, probing MYCN-amplified neuroblastoma as a model system, we showed that LIN28B expression was associated with enhanced cell migration in vitro and invasive and metastatic behavior in murine xenografts. Sequence analysis of the polyribosome fraction of LIN28B-expressing neuroblastoma cells revealed let-7-independent enrichment of transcripts encoding components of the translational and ribosomal apparatus and depletion of transcripts of neuronal developmental programs. We further observed that LIN28B utilizes both its cold shock and zinc finger RNA binding domains to preferentially interact with MYCN-induced transcripts of the ribosomal complex, enhancing their translation. These data demonstrated that LIN28B couples the MYCN-driven transcriptional program to enhanced ribosomal translation, thereby implicating LIN28B as a posttranscriptional driver of the metastatic phenotype.

Pegcrisantaspase in Combination with Venetoclax for Treatment of Relapsed or Refractory Acute Myeloid Leukemia: A Phase 1 Study

Background:Despite new therapeutic advances, acute myeloid leukemia (AML) still has poor outcomes, especially in patients with relapsed or refractory (R/R) disease with complex karyotype (CK) and/or TP53 mutation. Venetoclax (Ven), an oral BCL-2 inhibitor, in combination with DNA methyltransferase inhibitors (DNMTIs) has been approved by the FDA for treatment of newly diagnosed AML in adults who are unfit for intensive therapy with encouraging results, but the combination has been found to be less effective in patients with R/R AML.AML cells have been shown to be sensitive to extracellular glutamine depletion or manipulation of intracellular glutamine metabolism. Asparaginase converts asparagine and glutamine to aspartate and glutamate, decreasing plasma concentrations of asparagine and glutamine, with anti-leukemia activity. We previously published that crisantaspase produced complete plasma glutamine depletion in patients without dose-limiting toxicities and was associated with anti-leukemic activity in R/R AML (Emadi et al. Cancer Chemother Pharmacol 2018). In preclinical studies, we found that Pegcrisantaspase (PegC), a long-acting crisantaspase, not only had potent single-agent anti-AML activity, but also synergized with Ven in CK-AML cell lines and primary cells in vitro and in vivo (Emadi et al. Leukemia 2021). Ven-PegC targets the mTOR-eIF4E-driven ribosomal translational protein synthesis apparatus in AML. With no standardized treatment and poor outcomes for R/R AML, there is an unmet need for effective treatment options.Trial Design: We present an ongoing, non-randomized, open-label Phase 1 clinical trial evaluating Ven administered orally daily in combination with PegC administered intravenously every 14 days in 28-day treatment cycles in adults patients with R/R AML. The trial consists of two phases: dose escalation (four cohorts) and dose expansion at the final recommended phase 2 doses (RP2Ds).Adult patients with a pathologically confirmed diagnosis of AML whose disease has relapsed or is refractory to at least one line of AML therapy and with adequate organ function and no prior history of pancreatitis or ≥ Grade 3 thrombohemorrhagic events are eligible for this trial. All patients with FLT3, IDH1 or IDH2 mutation must have received at least one line of therapy with an available FLT3/IDH1/IDH2 inhibitor to be eligible for this trial. The study will include CK-AML and TP53-mutated AML.The primary objectives of the trial are to evaluate the safety and tolerability of Ven-PegC and estimate the maximum tolerated doses (MTDs) and/or biologically active doses (e.g. RP2D) of Ven-PegC in patients with R/R AML. The primary endpoints of the trial are incidences of regimen-limiting toxicities (RLTs) and treatment-emergent adverse events (TEAEs). The secondary endpoints include the rates of complete remission (CR) and composite complete remission (CR+CRh+CRi), event-free survival, overall survival, the rate of conversion from transfusion dependence to transfusion independence, and achievement of MRD <0.02% within 2 cycles of treatment with Ven-PegC. If a patient does not achieve at least hematologic improvement within 3 cycles of treatment, the patient will be taken off study. Responding patients can continue with the assigned doses until progression.The study uses a 3+3 design. Up to 24 subjects will be enrolled during dose escalation (in case exactly one RLT occurs in the first three patients enrolled at each of the four dose levels). Another 10 subjects will be enrolled at the final RP2D in an expansion cohort, for a total of 16 patients treated at the RP2D. The study is currently open at the University of Maryland Greenebaum Comprehensive Cancer Center. ClinicalTrials.gov Identifier is NCT04666649. [Display omitted] DisclosuresEmadi: Jazz Pharmaceuticals: Research Funding; NewLink Genetics: Research Funding; Servier: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Secura Bio.: Consultancy; KinaRx, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder.

Inhibition of Mitochondrial Translation By the Marine Natural Product Elatol Shows Potent Antileukemia Activity

Targeted signaling inhibitors for hematologic malignancies may lead to limited clinical efficacy due to the outgrowth of subpopulations with alternative pathways independent of the drug target. Relapse/refractory disease that results from treatment with targeted signaling inhibitors is a major hurdle in obtaining curative responses. Interestingly, work over the past decade or more has shown that chronic myelogenous leukemia (CML) stem cells (CD34+CD38-) are resistant to targeted signaling inhibitors, such as the BCR-ABL kinase class of inhibitors, often a problematic source of resistance leading to residual disease that may precipitate later progression (Hamilton et al., 2012).Recent studies have shown that some forms of lymphoma and leukemia cell have an energy metabolism highly dependent on mitochondrial oxidative phosphorylation (Ashton et al., 2018). Tigecycline, a US FDA approved antibiotic, has been shown to inhibit synthesis of mitochondrion-encoded proteins due to the similarity of bacterial and mitochondrial ribosomes, leading to selective lethality in hematologic malignancies reliant on enhanced oxidative phosphorylation (Norberg et al., 2017). Indeed, it was established that CML stem cells are reliant on upregulated oxidative phosphorylation, and combination treatment with the tyrosine-kinase inhibitor (TKI) imatinib and tigecycline eradicated therapy-resistant CML, both in vitro and in animal models (Kuntz et al., 2017).We have previously reported that elatol, the major compound from the red alga Laurencia microcladia, is effective against several non-Hodgkin lymphomas and primary chronic myelogenous leukemia cells (Peters et al., 2018). In vitro studies showed that elatol inhibits eIF4A1 helicase activity, suppressing cytoplasmic cap-dependent translation initiation. Further assessments using 35-S-methionine incorporation in HEK293T cells with or without single-digit micromolar concentrations of elatol for short time periods revealed strong downregulation of mitochondrion-encoded proteins as in Figure 1, (with no effect on mitochondrial transcription). This was confirmed in CML and acute lymphoblastic leukemia (ALL) cell lines whose 24-hour elatol LD50 ranged from high nanomolar to low micromolar concentrations. This potency was 10-40x higher than for tigecycline in side-by-side comparisons across several leukemia cell lines when compared at 72h. Additionally, we established that elatol does not affect integrity of small and large mitochondrial ribosomal units through sedimentation property analysis using sucrose gradients. Although the specific target on the mitochondrial translation apparatus remains elusive, we have uncovered that its mechanism of action differs from that of chloramphenicol, which inhibits translation elongation.In summary, we have performed proof-of-concept studies using HEK293T and HeLa cell lines, isolated mitochondria from HEK293T, and CML and ALL cell lines to reveal that elatol is a potent inhibitor of mitochondrial protein synthesis at concentrations that do not affect cytoplasmic protein synthesis and that this mechanism differs from chloramphenicol. Tigecycline's compelling preclinical data in combination with TKI informed design of a pending clinical trial (NCT02883036). Elatol's greatly improved potency provide a potential starting point for further optimization of this paradigm. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Broadening the Toolkit for Quantitatively Evaluating Noncanonical Amino Acid Incorporation in Yeast.

Genetic code expansion is a powerful approach for advancing critical fields such as biological therapeutic discovery. However, the machinery for genetically encoding noncanonical amino acids (ncAAs) is only available in limited plasmid formats, constraining potential applications. In extreme cases, the introduction of two separate plasmids, one containing an orthogonal translation system (OTS) to facilitate ncAA incorporation and a second for expressing a ncAA-containing protein of interest, is not possible due to a lack of the available selection markers. One strategy to circumvent this challenge is to express the OTS and protein of interest from a single vector. For what we believe is the first time in yeast, we describe here several sets of single plasmid systems (SPSs) for performing genetic code manipulation and compare the ncAA incorporation capabilities of these plasmids against the capabilities of previously described dual plasmid systems (DPSs). For both dual fluorescent protein reporters and yeast display reporters tested with multiple OTSs and ncAAs, measured ncAA incorporation efficiencies with SPSs were determined to be equal to efficiencies determined with DPSs. Click chemistry on yeast cells displaying ncAA-containing proteins was also shown to be feasible in both formats, although differences in reactivity between formats suggest the need for caution when using such approaches. Additionally, we investigated whether these reporters would support the separation of yeast strains known to exhibit distinct ncAA incorporation efficiencies. Model sorts conducted with mixtures of two strains transformed with the same SPS or DPS both led to the enrichment of a strain known to support a higher efficiency ncAA incorporation, suggesting that these reporters will be suitable for conducting screens for strains exhibiting enhanced ncAA incorporation efficiencies. Overall, our results confirm that SPSs are well behaved in yeast and provide a convenient alternative to DPSs. SPSs are expected to be invaluable for conducting high-throughput investigations of the effects of genetic or genomic changes on ncAA incorporation efficiency and, more fundamentally, the eukaryotic translation apparatus.

The Candida glabrata Upc2A transcription factor is a global regulator of antifungal drug resistance pathways.

The most commonly used antifungal drugs are the azole compounds, which interfere with biosynthesis of the fungal-specific sterol: ergosterol. The pathogenic yeast Candida glabrata commonly acquires resistance to azole drugs like fluconazole via mutations in a gene encoding a transcription factor called PDR1. These PDR1 mutations lead to overproduction of drug transporter proteins like the ATP-binding cassette transporter Cdr1. In other Candida species, mutant forms of a transcription factor called Upc2 are associated with azole resistance, owing to the important role of this protein in control of expression of genes encoding enzymes involved in the ergosterol biosynthetic pathway. Recently, the C. glabrata Upc2A factor was demonstrated to be required for normal azole resistance, even in the presence of a hyperactive mutant form of PDR1. Using genome-scale approaches, we define the network of genes bound and regulated by Upc2A. By analogy to a previously described hyperactive UPC2 mutation found in Saccharomyces cerevisiae, we generated a similar form of Upc2A in C. glabrata called G898D Upc2A. Analysis of Upc2A genomic binding sites demonstrated that wild-type Upc2A binding to target genes was strongly induced by fluconazole while G898D Upc2A bound similarly, irrespective of drug treatment. Transcriptomic analyses revealed that, in addition to the well-described ERG genes, a large group of genes encoding components of the translational apparatus along with membrane proteins were responsive to Upc2A. These Upc2A-regulated membrane protein-encoding genes are often targets of the Pdr1 transcription factor, demonstrating the high degree of overlap between these two regulatory networks. Finally, we provide evidence that Upc2A impacts the Pdr1-Cdr1 system and also modulates resistance to caspofungin. These studies provide a new perspective of Upc2A as a master regulator of lipid and membrane protein biosynthesis.

Incomplete tricarboxylic acid cycle and proton gradient in Pandoravirus massiliensis: is it still a virus?

The discovery of Acanthamoeba polyphaga Mimivirus, the first isolated giant virus of amoeba, challenged the historical hallmarks defining a virus. Giant virion sizes are known to reach up to 2.3 µm, making them visible by optical microscopy. Their large genome sizes of up to 2.5 Mb can encode proteins involved in the translation apparatus. We have investigated possible energy production in Pandoravirus massiliensis. Mitochondrial membrane markers allowed for the detection of a membrane potential in purified virions and this was enhanced by a regulator of the tricarboxylic acid cycle but abolished by the use of a depolarizing agent. Bioinformatics was employed to identify enzymes involved in virion proton gradient generation and this approach revealed that eight putative P. massiliensis proteins exhibited low sequence identities with known cellular enzymes involved in the universal tricarboxylic acid cycle. Further, all eight viral genes were transcribed during replication. The product of one of these genes, ORF132, was cloned and expressed in Escherichia coli, and shown to function as an isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle. Our findings show for the first time that a membrane potential can exist in Pandoraviruses, and this may be related to tricarboxylic acid cycle. The presence of a proton gradient in P. massiliensis makes this virus a form of life for which it is legitimate to ask the question “what is a virus?”.

Noroviruses-The State of the Art, Nearly Fifty Years after Their Initial Discovery.

Human noroviruses are recognised as the major global cause of viral gastroenteritis. Here, we provide an overview of notable advances in norovirus research and provide a short recap of the novel model systems to which much of the recent progress is owed. Significant advances include an updated classification system, the description of alternative virus-like protein morphologies and capsid dynamics, and the further elucidation of the functions and roles of various viral proteins. Important milestones include new insights into cell tropism, host and microbial attachment factors and receptors, interactions with the cellular translational apparatus, and viral egress from cells. Noroviruses have been detected in previously unrecognised hosts and detection itself is facilitated by improved analytical techniques. New potential transmission routes and/or viral reservoirs have been proposed. Recent in vivo and in vitro findings have added to the understanding of host immunity in response to norovirus infection, and vaccine development has progressed to preclinical and even clinical trial testing. Ongoing development of therapeutics includes promising direct-acting small molecules and host-factor drugs.

Not4 and Not5 modulate translation elongation by Rps7A ubiquitination, Rli1 moonlighting, and condensates that exclude eIF5A.

In this work, we show that Not4 and Not5 from the Ccr4-Not complex modulate translation elongation dynamics and change ribosome A-site dwelling occupancy in a codon-dependent fashion. These codon-specific changes in not5Δ cells are very robust and independent of codon position within the mRNA, the overall mRNA codon composition, or changes of mRNA expression levels. They inversely correlate with codon-specific changes in cells depleted for eIF5A and positively correlate with those in cells depleted for ribosome-recycling factor Rli1. Not5 resides in punctate loci, co-purifies with ribosomes and Rli1, but not with eIF5A, and limits mRNA solubility. Overexpression of wild-type or non-complementing Rli1 and loss of Rps7A ubiquitination enable Not4 E3 ligase-dependent translation of polyarginine stretches. We propose that Not4 and Not5 modulate translation elongation dynamics to produce a soluble proteome by Rps7A ubiquitination, dynamic condensates that limit mRNA solubility and exclude eIF5A, and a moonlighting function of Rli1.