Understanding functions of eEF1 translation elongation factors beyond translation. A proteomic approach.

Mammalian translation elongation factors eEF1A1 and eEF1A2 are 92% homologous isoforms whose mutually exclusive tissue-specific expression is regulated during development. The isoforms have similar translation functionality, but show differences in spatial organization and participation in various processes, such as oncogenesis and virus reproduction. The differences may be due to their ability to interact with isoform-specific partner proteins. We used the identified sets of eEF1A1 or eEF1A2 partner proteins to identify cell complexes and/or processes specific to one particular isoform. As a result, we found isoform-specific interactions reflecting the involvement of different eEF1A isoforms in different cellular processes, including actin-related, chromatin-remodeling, ribonuclease H2, adenylyl cyclase, and Cul3-RING ubiquitin ligase complexes as well as initiation of mitochondrial transcription. An essential by-product of our analysis is the elucidation of a number of cellular processes beyond protein biosynthesis, where both isoforms appear to participate such as large ribosomal subunit biogenesis, mRNA splicing, DNA mismatch repair, 26S proteasome activity, P-body and exosomes formation, protein targeting to the membrane. This information suggests that a relatively high content of eEF1A in the cell may be necessary not only to maintain efficient translation, but also to ensure its participation in various cellular processes, where some roles of eEF1A have not yet been described. We believe that the data presented here will be useful for deciphering new auxiliary functions of eEF1A and its isoforms, and provide a new look at the known non-canonical functions of this main component of the human translation-elongation machinery.

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.

Structural dynamics of translation elongation factor Tu during aa-tRNA delivery to the ribosome.

The GTPase elongation factor EF-Tu delivers aminoacyl-tRNAs to the mRNA-programmed ribosome during translation. Cognate codon-anticodon interaction stimulates GTP hydrolysis within EF-Tu. It has been proposed that EF-Tu undergoes a large conformational change subsequent to GTP hydrolysis, which results in the accommodation of aminoacyl-tRNA into the ribosomal A-site. However, this proposal has never been tested directly. Here, we apply single-molecule total internal reflection fluorescence microscopy to study the conformational dynamics of EF-Tu when bound to the ribosome. Our studies show that GTP hydrolysis initiates a partial, comparatively small conformational change of EF-Tu on the ribosome, not directly along the path from the solution 'GTP' to the 'GDP' structure. The final motion is completed either concomitant with or following dissociation of EF-Tu from the ribosome. The structural transition of EF-Tu on the ribosome is slower when aa-tRNA binds to a cognate versus a near-cognate codon. The resulting longer residence time of EF-Tu on the ribosome may be important for promoting accommodation of the cognate aminoacyl-tRNA into the A-site.

E. coli elongation factor Tu bound to a GTP analogue displays an open conformation equivalent to the GDP-bound form.

According to the traditional view, GTPases act as molecular switches, which cycle between distinct 'on' and 'off' conformations bound to GTP and GDP, respectively. Translation elongation factor EF-Tu is a GTPase essential for prokaryotic protein synthesis. In its GTP-bound form, EF-Tu delivers aminoacylated tRNAs to the ribosome as a ternary complex. GTP hydrolysis is thought to cause the release of EF-Tu from aminoacyl-tRNA and the ribosome due to a dramatic conformational change following Pi release. Here, the crystal structure of Escherichia coli EF-Tu in complex with a non-hydrolysable GTP analogue (GDPNP) has been determined. Remarkably, the overall conformation of EF-Tu·GDPNP displays the classical, open GDP-bound conformation. This is in accordance with an emerging view that the identity of the bound guanine nucleotide is not 'locking' the GTPase in a fixed conformation. Using a single-molecule approach, the conformational dynamics of various ligand-bound forms of EF-Tu were probed in solution by fluorescence resonance energy transfer. The results suggest that EF-Tu, free in solution, may sample a wider set of conformations than the structurally well-defined GTP- and GDP-forms known from previous X-ray crystallographic studies. Only upon binding, as a ternary complex, to the mRNA-programmed ribosome, is the well-known, closed GTP-bound conformation, observed.

Dynamics of P-type ATPase transport revealed by single-molecule FRET.

Phosphorylation-type (P-type) ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest that the transport mechanism is defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have used single-molecule fluorescence resonance energy transfer to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca2+-ATPase (LMCA1), an orthologue of eukaryotic Ca2+-ATPases. We identify key intermediates with no known crystal structures and show that Ca2+ efflux by LMCA1 is rate-limited by phosphoenzyme formation. The transport process involves reversible steps and an irreversible step that follows release of ADP and extracellular release of Ca2+.

Living with an implantable cardioverter defibrillator: patients' preferences and needs for information provision and care options.

AIMS: The clinical management and care of patients with an implantable cardioverter defibrillator (ICD) has shifted from face-to-face in-clinic visits to remote monitoring. Reduced interactions between patients and healthcare professionals may impede patients' transition to adapting post-implant. We examined patients' needs and preferences for information provision and care options and overall satisfaction with treatment. METHODS AND RESULTS: Patients implanted with a first-time ICD or defibrillator with cardiac resynchronization therapy (n = 389) within the last 2 years at Odense University Hospital were asked to complete a purpose-designed and standardized set of questionnaires. The level of satisfaction with information provision was high; only 13.1% were dissatisfied. Psychological support for patients (39.9%), their relatives (43.1%), and deactivation of the ICD towards end of life (47.8%) were among the top five topics that patients reported to have received no information about. The top five care options that patients had missed were talking to the same healthcare professional (75.2%), receiving ongoing feedback via remote monitoring (61.1%), having a personal conversation with a staff member 2-3 weeks post-implant (59.6%), having an exercise tolerance test (52.5%), and staff asking how patients felt while hospitalized (50.4%). Patients with a secondary prevention indication and cardiac arrest survivors had specific needs, including a wish for a psychological consult post-discharge. CONCLUSION: Despite a high satisfaction level with information provision, particular topics are not broached with patients (e.g. device activation) and patients have unmet needs that are not met in current clinical practice.

Engineering a Prototypic P-type ATPase Listeria monocytogenes Ca(2+)-ATPase 1 for Single-Molecule FRET Studies.

Approximately 30% of the ATP generated in the living cell is utilized by P-type ATPase primary active transporters to generate and maintain electrochemical gradients across biological membranes. P-type ATPases undergo large conformational changes during their functional cycle to couple ATP hydrolysis in the cytoplasmic domains to ion transport across the membrane. The Ca(2+)-ATPase from Listeria monocytogenes, LMCA1, was found to be a suitable model of P-type ATPases and was engineered to facilitate single-molecule FRET studies of transport-related structural changes. Mutational analyses of the endogenous cysteine residues in LMCA1 were performed to reduce background labeling without compromising activity. Pairs of cysteines were introduced into the optimized low-reactivity background, and labeled with maleimide derivatives of Cy3 and Cy5 resulting in site-specifically double-labeled protein with moderate activity. Ensemble and confocal single-molecule FRET studies revealed changes in FRET distribution related to structural changes during the transport cycle, consistent with those observed by X-ray crystallography for the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA). Notably, the cytosolic headpiece of LMCA1 was found to be distinctly more compact in the E1 state than in the E2 state. Thus, the established experimental system should allow future real-time FRET studies of the structural dynamics of LMCA1 as a representative P-type ATPase.

Study Design and Cohort Description of DEFIB-WOMEN: A National Danish Study in Patients with an ICD.

BACKGROUND: Little systematic evidence is available on potential gender differences in patients with an implantable cardioverter defibrillator (ICD) from a real-world cohort. We designed the DEFIB-WOMEN (The Utilization of Implantable Cardioverter DEFIBrillator Therapy in the Treatment of Heart Disease: Clinical and Psychological outcomes in WOMEN) study to examine gender differences on (1) patient-reported outcomes (PROs), (2) procedure- and device-related complications, and (3) ventricular tachyarrhythmia and mortality. This presents the study design and baseline characteristics of the cohort. METHODS: DEFIB-WOMEN is a national, multicenter, prospective, observational study. First-time implanted patients are asked to complete PROs at several time points. Information on baseline and follow-up characteristics are captured from patients' medical records, purpose-designed questions, and the Danish national registers. The DEFIB-WOMEN cohort is composed of 1,790 (19% women; 343/1,790) patients implanted between June 2010 and April 2013. RESULTS: Women and men differed on several demographic and clinical baseline characteristics, including on the prescription of ß-blockers, statins, angiotensin-converting enzyme inhibitors, and psychotropic agents. Although women generally had a healthier clinical profile, they reported significantly more symptoms of anxiety and depression and ICD concerns (fear of shock) as compared to men. These differences were not only statistically significant but also clinically relevant, with the magnitude of the differences in anxiety and ICD concerns being 0.44 and 0.42, respectively, as indicated by Cohen's effect size index. CONCLUSIONS: These preliminary results indicate that women with an ICD experience particularly more anxiety and ICD concerns as compared to men at the time of implant. Future results of DEFIB-WOMEN will show whether these gender differences persist and whether there are also gender differences in complications and survival.

Structural basis for RNA-genome recognition during bacteriophage Qß replication.

Upon infection of Escherichia coli by bacteriophage Qß, the virus-encoded ß-subunit recruits host translation elongation factors EF-Tu and EF-Ts and ribosomal protein S1 to form the Qß replicase holoenzyme complex, which is responsible for amplifying the Qß (+)-RNA genome. Here, we use X-ray crystallography, NMR spectroscopy, as well as sequence conservation, surface electrostatic potential and mutational analyses to decipher the roles of the ß-subunit and the first two oligonucleotide-oligosaccharide-binding domains of S1 (OB1-2) in the recognition of Qß (+)-RNA by the Qß replicase complex. We show how three basic residues of the ß subunit form a patch located adjacent to the OB2 domain, and use NMR spectroscopy to demonstrate for the first time that OB2 is able to interact with RNA. Neutralization of the basic residues by mutagenesis results in a loss of both the phage infectivity in vivo and the ability of Qß replicase to amplify the genomic RNA in vitro. In contrast, replication of smaller replicable RNAs is not affected. Taken together, our data suggest that the ß-subunit and protein S1 cooperatively bind the (+)-stranded Qß genome during replication initiation and provide a foundation for understanding template discrimination during replication initiation.

Two rare deletions upstream of the NRXN1 gene (2p16.3) affecting the non-coding mRNA AK127244 segregate with diverse psychopathological phenotypes in a family.

CNVs spanning the 2p16.3 (NRXN1) and the 15q11.2 gene rich region have been associated with severe neuropsychiatric disorders including schizophrenia. Recently, studies have also revealed that CNVs in non-coding regions play an essential role in genomic variability in addition to disease susceptibility. In this study, we describe a family affected by a wide range of psychiatric disorders including early onset schizophrenia, schizophreniform disorder, and affective disorders. Microarray analysis identified two rare deletions immediately upstream of the NRXN1 gene affecting the non-coding mRNA AK127244 in addition to the pathogenic 15q11.2 deletion in distinct family members. The two deletions upstream of the NRXN1 gene were found to segregate with psychiatric disorders in the family and further similar deletions have been observed in patients diagnosed with autism spectrum disorder. Thus, we suggest that non-coding regions upstream of the NRXN1 gene affecting AK127244 might (as NRXN1) contain susceptibility regions for a wide spectrum of neuropsychiatric disorders.

EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways.

The G-protein EF-Tu, which undergoes a major conformational change when EF-Tu·GTP is converted to EF-Tu·GDP, forms part of an aminoacyl(aa)-tRNA·EF-Tu·GTP ternary complex (TC) that accelerates the binding of aa-tRNA to the ribosome during peptide elongation. Such binding, placing a portion of EF-Tu in contact with the GTPase Associated Center (GAC), is followed by GTP hydrolysis and Pi release, and results in formation of a pretranslocation (PRE) complex. Although tRNA movement through the ribosome during PRE complex formation has been extensively studied, comparatively little is known about the dynamics of EF-Tu interaction with either the ribosome or aa-tRNA. Here we examine these dynamics, utilizing ensemble and single molecule assays employing fluorescent labeled derivatives of EF-Tu, tRNA, and the ribosome to measure changes in either FRET efficiency or fluorescence intensity during PRE complex formation. Our results indicate that ribosome-bound EF-Tu separates from the GAC prior to its full separation from aa-tRNA, and suggest that EF-Tu·GDP dissociates from the ribosome by two different pathways. These pathways correspond to either reversible EF-Tu·GDP dissociation from the ribosome prior to the major conformational change in EF-Tu that follows GTP hydrolysis, or irreversible dissociation after or concomitant with this conformational change.

Structural outline of the detailed mechanism for elongation factor Ts-mediated guanine nucleotide exchange on elongation factor Tu.

Translation elongation factor EF-Tu belongs to the superfamily of guanine-nucleotide binding proteins, which play key cellular roles as regulatory switches. All G-proteins require activation via exchange of GDP for GTP to carry out their respective tasks. Often, guanine-nucleotide exchange factors are essential to this process. During translation, EF-Tu:GTP transports aminoacylated tRNA to the ribosome. GTP is hydrolyzed during this process, and subsequent reactivation of EF-Tu is catalyzed by EF-Ts. The reaction path of guanine-nucleotide exchange is structurally poorly defined for EF-Tu and EF-Ts. We have determined the crystal structures of the following reaction intermediates: two structures of EF-Tu:GDP:EF-Ts (2.2 and 1.8Å resolution), EF-Tu:PO4:EF-Ts (1.9Å resolution), EF-Tu:GDPNP:EF-Ts (2.2Å resolution) and EF-Tu:GDPNP:pulvomycin:Mg(2+):EF-Ts (3.5Å resolution). These structures provide snapshots throughout the entire exchange reaction and suggest a mechanism for the release of EF-Tu in its GTP conformation. An inferred sequence of events during the exchange reaction is presented.

Labeled EF-Tus for rapid kinetic studies of pretranslocation complex formation.

The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics.

An eEF1A1 truncation encoded by PTI-1 exerts its oncogenic effect inside the nucleus.

BACKGROUND: The oncogene PTI-1 was originally isolated from a prostate cancer cell line by its capability to transform rat fibroblasts. The PTI-1 mRNA has a very eccentric structure as the 5'UTR is similar to prokaryotic 23S rRNA, while the major open reading frame and the 3'UTR corresponds to a part of the mRNA encoding human translation elongation factor eEF1A1. Thus, the largest open reading frame encodes a truncated version of eEF1A1 lacking the first 67 amino acids, while having three unique N-terminal amino acids. Previously, the UTRs were shown to be a prerequisite for the transforming capacity of the PTI-1 transcript. In this study, we have investigated the possible role of the UTRs in regulating protein expression and localization. METHODS: The protein expression profiles of a number of PTI-1 mRNA variants were studied in vitro and in vivo. Furthermore, the oncogenic potentials of the same PTI-1 mRNAs were determined by monitoring the capacities of stably transfected cells expressing these mRNAs to induce tumors in nude mice and form foci in cell culture. Finally, the cellular localizations of PTI-1 proteins expressed from these mRNAs were determined by fluorescence microscopy. RESULTS: The PTI-1 mRNA was found to give rise to multiple protein products that potentially originate from translation initiation at downstream, inframe AUGs within the major open reading frame. At least one of the truncated protein variants was also found to be oncogenic. However, the UTRs did not appear to influence the amount and identities of these truncated protein products. In contrast, our localization studies showed that the UTRs of the transcript promote a nuclear localization of the encoded protein(s). CONCLUSIONS: Translation of the PTI-1 mRNA results in multiple protein products of which (a) truncated variant(s) may play a predominant role during cellular transformation. The PTI-1 UTRs did not seem to play a role in translation regulation, but appeared to contribute to a nuclear localization of the PTI-1 protein(s). This indicates that the PTI-1 protein(s) exert(s) its/their oncogenic function inside the nucleus.

The busiest of all ribosomal assistants: elongation factor Tu.

During translation, the nucleic acid language employed by genes is translated into the amino acid language used by proteins. The translator is the ribosome, while the dictionary employed is known as the genetic code. The genetic information is presented to the ribosome in the form of a mRNA, and tRNAs connect the two languages. Translation takes place in three steps: initiation, elongation, and termination. After a protein has been synthesized, the components of the translation apparatus are recycled. During each phase of translation, the ribosome collaborates with specific translation factors, which secure a proper balance between speed and fidelity. Notably, initiation, termination, and ribosomal recycling occur only once per protein produced during normal translation, while the elongation step is repeated a large number of times, corresponding to the number of amino acids constituting the protein of interest. In bacteria, elongation factor Tu plays a central role during the selection of the correct amino acids throughout the elongation phase of translation. Elongation factor Tu is the main subject of this review.

Identification of an mRNP complex regulating tumorigenesis at the translational elongation step.

Transcript-selective translational regulation of epithelial-mesenchymal transition (EMT) by transforming growth factor-ß (TGF-ß) is directed by the hnRNP E1-containing TGF-ß-activated-translational (BAT) mRNP complex. Herein, eukaryotic elongation factor-1 A1 (eEF1A1) is identified as an integral component of the BAT complex. Translational silencing of Dab2 and ILEI, two EMT transcripts, is mediated by the binding of hnRNP E1 and eEF1A1 to their 3'UTR BAT element, whereby hnRNP E1 stalls translational elongation by inhibiting the release of eEF1A1 from the ribosomal A site. TGF-ß-mediated hnRNP E1 phosphorylation, through Akt2, disrupts the BAT complex, thereby restoring translation of target EMT transcripts. Attenuation of hnRNP E1 expression in two noninvasive breast epithelial cells (NMuMG and MCF-7) not only induced EMT but also enabled cells to form metastatic lesions in vivo. Thus, translational regulation by TGF-ß at the elongation stage represents a critical checkpoint coordinating the expression of EMT transcripts required during development and in tumorigenesis and metastatic progression.

Aminoacyl-tRNA-charged eukaryotic elongation factor 1A is the bona fide substrate for Legionella pneumophila effector glucosyltransferases.

Legionella pneumophila, which is the causative organism of Legionnaires disease, translocates numerous effector proteins into the host cell cytosol by a type IV secretion system during infection. Among the most potent effector proteins of Legionella are glucosyltransferases (lgt's), which selectively modify eukaryotic elongation factor (eEF) 1A at Ser-53 in the GTP binding domain. Glucosylation results in inhibition of protein synthesis. Here we show that in vitro glucosylation of yeast and mouse eEF1A by Lgt3 in the presence of the factors Phe-tRNA(Phe) and GTP was enhanced 150 and 590-fold, respectively. The glucosylation of eEF1A catalyzed by Lgt1 and 2 was increased about 70-fold. By comparison of uncharged tRNA with two distinct aminoacyl-tRNAs (His-tRNA(His) and Phe-tRNA(Phe)) we could show that aminoacylation is crucial for Lgt-catalyzed glucosylation. Aminoacyl-tRNA had no effect on the enzymatic properties of lgt's and did not enhance the glucosylation rate of eEF1A truncation mutants, consisting of the GTPase domain only or of a 5 kDa peptide covering Ser-53 of eEF1A. Furthermore, binding of aminoacyl-tRNA to eEF1A was not altered by glucosylation. Taken together, our data suggest that the ternary complex, consisting of eEF1A, aminoacyl-tRNA and GTP, is the bona fide substrate for lgt's.

Structure of the Qbeta replicase, an RNA-dependent RNA polymerase consisting of viral and host proteins.

The RNA-dependent RNA polymerase core complex formed upon infection of Escherichia coli by the bacteriophage Qbeta is composed of the viral catalytic beta-subunit as well as the host translation elongation factors EF-Tu and EF-Ts, which are required for initiation of RNA replication. We have determined the crystal structure of the complex between the beta-subunit and the two host proteins to 2.5-A resolution. Whereas the basic catalytic machinery in the viral subunit appears similar to other RNA-dependent RNA polymerases, a unique C-terminal region of the beta-subunit engages in extensive interactions with EF-Tu and may contribute to the separation of the transient duplex formed between the template and the nascent product to allow exponential amplification of the phage genome. The evolution of resistance by the host appears to be impaired because of the interactions of the beta-subunit with parts of EF-Tu essential in recognition of aminoacyl-tRNA.

Properties of Escherichia coli EF-Tu mutants designed for fluorescence resonance energy transfer from tRNA molecules.

Here we describe the design, preparation and characterization of 10 EF-Tu mutants of potential utility for the study of Escherichia coli elongation factor Tu (EF-Tu) interaction with tRNA by a fluorescence resonance energy transfer assay. Each mutant contains a single cysteine residue at positions in EF-Tu that are proximal to tRNA sites within the aminoacyl-tRNA.EF-Tu.GTP ternary complex that have previously been labeled with fluorophores. These positions fall in the 323-326 and 344-348 regions of EF-Tu, and at the C terminus. The EF-Tus were isolated as N-terminal fusions to glutathione S-transferase (GST), which was cleaved to yield intact EF-Tus. The mutant EF-Tus were tested for binding to GDP, binding to tRNA in gel retardation and protection assays, and activity in poly-U translation in vitro. The results indicate that at least three EF-Tu mutants, K324C, G325C and E348C, are suitable for further studies. Remarkably, GST fusions that were not cleaved were also active in the various assays, despite the N-terminal fusion.

Translation elongation factor eEF1A binds to a novel myosin binding protein-C-like protein.

Eukaryotic translation elongation factor 1A (eEF1A) is a guanine-nucleotide binding protein, which transports aminoacylated tRNA to the ribosomal A site during protein synthesis. In a yeast two-hybrid screening of a human skeletal muscle cDNA library, a novel eEF1A binding protein, immunoglobulin-like and fibronectin type III domain containing 1 (IGFN1), was discovered, and its interaction with eEF1A was confirmed in vitro. IGFN1 is specifically expressed in skeletal muscle and presents immunoglobulin I and fibronectin III sets of domains characteristic of sarcomeric proteins. IGFN1 shows sequence and structural homology to myosin binding protein-C fast and slow-type skeletal muscle isoforms. IGFN1 is substantially upregulated during muscle denervation. We propose a model in which this increased expression of IGFN1 serves to down-regulate protein synthesis via interaction with eEF1A during denervation.