The eukaryotic initiation factor 5A (eIF5A1), the molecule, mechanisms and recent insights into the pathophysiological roles

Since the demonstration of its involvement in cell proliferation, the eukaryotic initiation factor 5A (eIF5A) has been studied principally in relation to the development and progression of cancers in which the isoform A2 is mainly expressed. However, an increasing number of studies report that the isoform A1, which is ubiquitously expressed in normal cells, exhibits novel molecular features that reveal its new relationships between cellular functions and organ homeostasis. At a first glance, eIF5A can be regarded, among other things, as a factor implicated in the initiation of translation. Nevertheless, at least three specificities: (1) its extreme conservation between species, including plants, throughout evolution, (2) its very special and unique post-translational modification through the activating-hypusination process, and finally (3) its close relationship with the polyamine pathway, suggest that the role of eIF5A in living beings remains to be uncovered. In fact, and beyond its involvement in facilitating the translation of proteins containing polyproline residues, eIF5A is implicated in various physiological processes including ischemic tolerance, metabolic adaptation, aging, development, and immune cell differentiation. These newly discovered physiological properties open up huge opportunities in the clinic for pathologies such as, for example, the ones in which the oxygen supply is disrupted. In this latter case, organ transplantation, myocardial infarction or stroke are concerned, and the current literature defines eIF5A as a new drug target with a high level of potential benefit for patients with these diseases or injuries. Moreover, the recent use of genomic and transcriptomic association along with metadata studies also revealed the implication of eIF5A in genetic diseases. Thus, this review provides an overview of eIF5A from its molecular mechanism of action to its physiological roles and the clinical possibilities that have been recently reported in the literature.

Elucidation of the Translation Initiation Factor Interaction Network of Haloferax volcanii Reveals Coupling of Transcription and Translation in Haloarchaea

Translation is an important step in gene expression. Initiation of translation is rate-limiting, and it is phylogenetically more diverse than elongation or termination. Bacteria contain only three initiation factors. In stark contrast, eukaryotes contain more than 10 (subunits of) initiation factors (eIFs). The genomes of archaea contain many genes that are annotated to encode archaeal homologs of eukaryotic initiation factors (aIFs). However, experimental characterization of aIFs is scarce and mostly restricted to very few species. To broaden the view, the protein–protein interaction network of aIFs in the halophilic archaeon Haloferax volcanii has been characterized. To this end, tagged versions of 14 aIFs were overproduced, affinity isolated, and the co-isolated binding partners were identified by peptide mass fingerprinting and MS/MS analyses. The aIF–aIF interaction network was resolved, and it was found to contain two interaction hubs, (1) the universally conserved factor aIF5B, and (2) a protein that has been annotated as the enzyme ribose-1,5-bisphosphate isomerase, which we propose to rename to aIF2Bα. Affinity isolation of aIFs also led to the co-isolation of many ribosomal proteins, but also transcription factors and subunits of the RNA polymerase (Rpo). To analyze a possible coupling of transcription and translation, seven tagged Rpo subunits were overproduced, affinity isolated, and co-isolated proteins were identified. The Rpo interaction network contained many transcription factors, but also many ribosomal proteins as well as the initiation factors aIF5B and aIF2Bα. These results showed that transcription and translation are coupled in haloarchaea, like in Escherichia coli. It seems that aIF5B and aIF2Bα are not only interaction hubs in the translation initiation network, but also key players in the transcription-translation coupling.

Protein Nucleotidylylation in +ssRNA Viruses

Nucleotidylylation is a post-transcriptional modification important for replication in the picornavirus supergroup of RNA viruses, including members of the Caliciviridae, Coronaviridae, Picornaviridae and Potyviridae virus families. This modification occurs when the RNA-dependent RNA polymerase (RdRp) attaches one or more nucleotides to a target protein through a nucleotidyl-transferase reaction. The most characterized nucleotidylylation target is VPg (viral protein genome-linked), a protein linked to the 5′ end of the genome in Caliciviridae, Picornaviridae and Potyviridae. The nucleotidylylation of VPg by RdRp is a critical step for the VPg protein to act as a primer for genome replication and, in Caliciviridae and Potyviridae, for the initiation of translation. In contrast, Coronaviridae do not express a VPg protein, but the nucleotidylylation of proteins involved in replication initiation is critical for genome replication. Furthermore, the RdRp proteins of the viruses that perform nucleotidylylation are themselves nucleotidylylated, and in the case of coronavirus, this has been shown to be essential for viral replication. This review focuses on nucleotidylylation within the picornavirus supergroup of viruses, including the proteins that are modified, what is known about the nucleotidylylation process and the roles that these modifications have in the viral life cycle.

Free energy landscape of RNA binding dynamics in start codon recognition by eukaryotic ribosomal pre-initiation complex

Specific interaction between the start codon, 5’-AUG-3’, and the anticodon, 5’-CAU-3’, ensures accurate initiation of translation. Recent studies show that several near-cognate start codons (e.g. GUG and CUG) can play a role in initiating translation in eukaryotes. However, the mechanism allowing initiation through mismatched base-pairs at the ribosomal decoding site is still unclear at an atomic level. In this work, we propose an extended simulation-based method to evaluate free energy profiles, through computing the distance between each base-pair of the triplet interactions involved in recognition of start codons in eukaryotic translation pre-initiation complex. Our method provides not only the free energy penalty for mismatched start codons relative to the AUG start codon, but also the preferred pathways of transitions between bound and unbound states, which has not been described by previous studies. To verify the method, the binding dynamics of cognate (AUG) and near-cognate start codons (CUG and GUG) were simulated. Evaluated free energy profiles agree with experimentally observed changes in initiation frequencies from respective codons. This work proposes for the first time how a G:U mismatch at the first position of codon (GUG)-anticodon base-pairs destabilizes the accommodation in the initiating eukaryotic ribosome and how initiation at a CUG codon is nearly as strong as, or sometimes stronger than, that at a GUG codon. Our method is expected to be applied to study the affinity changes for various mismatched base-pairs.

Lower Synthesis and Higher Catabolism of Liver and Muscle Protein Compensate for Amino Acid Deficiency in Severely Protein-Restricted Growing Rat

Objectives Severely low-protein (LP) diets induce a decrease in body weight and an increase in relative food including intake (FI) in rat. In the liver, changes in anabolic and catabolic protein pathways could transitorily participate to compensate for amino acid (AA) deficiency. The present study investigated these liver and muscle protein metabolic pathways on LP diet fed growing rats. Methods Growing rats were fed for three weeks different diets containing 3–5–8–12–15 or 20% energy from milk protein. Body weight and FI were measured daily. At the end of the experiment, rats were injected with 13C valine and tissues and biological fluids were collected for gene expression measurement, blood AA UPLC analysis and protein synthesis rate determination in liver and muscle. Statistical analysis was done by 1- or 2-factor ANOVA, when data were repeated. Results P3, P5 and P8% diets resulted in significant growth retardation and significant decrease in lean mass. Severe protein deficiency induced a decrease in the rate of protein synthesis in the liver and muscle. In addition, the results showed activation of the GCN2 pathway, via ATF4-CHOP-TRB3 both in the liver and in the muscle, which suggests the inhibition of the initiation of translation at the level of the binding of the RNAt-Met. Liver proteolytic pathways were up-regulated including the ubiquitin-proteasome, the caspase system and the autophagy. In muscle, both the ubiquitin-proteasome pathway, and autophagy were increased as well as the calpain system. The GCN2 pathway, via ATF4-CHOP-TRB3 was activated in both liver and muscle, confirming the activation of protein degradation by the ubiquitin-proteasome pathways, and autophagy. In portal vein, indispensable AA were lower in severe protein deficient diet whereas in vena cava no difference was observed. Conclusions Severe protein restriction lowered protein synthesis and activated protein catabolism in both liver and muscle whereas no effect was observed for moderate protein restriction. These results confirm that the liver and muscle play a major role in supplying the body with indispensable AA in response to severe protein restriction. Funding Sources This study was funded by the doctoral school ABIES and AlimH-INRAE department.

Identification of Cryptic Putative IRESs Initiating the Translation of Nonstructural Proteins Encoded by the HRV16 Genome

Cap-dependent initiation of translation is a canonical mechanism adopted by eukaryotic cells. Internal ribosome entry site (IRES)-dependent translation is a mechanism distinct from 5′ cap-dependent translation. IRES elements are located mainly in the 5′-untranslated regions (UTRs) of viral and eukaryotic mRNAs. In addition, IRESs are found in the coding regions of some viral and eukaryotic genomes and initiate the translation of some functional truncated isoforms. Here, via IRES-initiated expression of proteins, bicistronic vectors and ribosome profiling of the human rhinovirus 16 (HRV16), we found that the coding region of the nonstructural proteins P2 and P3 contained 5 putative IRES elements. These 5 putative IRESs were located within nucleotides 4286-4585, 5002-5126, 6245-6394, 6619-6718 and 6629-6778 and initiated green fluorescent protein (GFP) expression in vitro. This alternative mechanism might be effective and economical for eliminating the time and raw material required to synthesize the full-length polyprotein.

Free Energy Landscape of RNA Binding Dynamics in Start Codon Recognition by Eukaryotic Ribosomal Pre-Initiation Complex

Specific interaction between the start codon, 5’-AUG-3’, and the anticodon, 5’-CAU-3’, ensures accurate initiation of translation. Recent studies show that several near-cognate start codons (e.g. GUG and CUG) can play a role in initiating translation in eukaryotes. However, the mechanism allowing initiation through mismatched base-pairs at the ribosomal decoding site is still unclear at an atomic level. In this work, we propose an extended simulation-based method to evaluate free energy profiles, through computing the distance between each base-pair of the triplet interactions (d1, d2 and d3) involved in recognition of start codons in eukaryotic translation pre-initiation complex. Our method provides not only the free energy penalty (ΔΔG) for mismatched start codons relative to the AUG start codon, but also the preferred pathways of transitions between bound and unbound states, which has not been described by previous studies. To verify the method, the binding dynamics of cognate (AUG) and near-cognate start codons (CUG and GUG) were simulated. Evaluated free energy profiles agree with experimentally observed changes in initiation frequencies from respective codons. This work proposes for the first time how a G:U mismatch at the first position of codon (GUG)-anticodon base-pairs destabilizes the accommodation in the initiating eukaryotic ribosome and how initiation at a CUG codon is nearly as strong as, or sometimes stronger than, that at a GUG codon. Our method is expected to be applied to study the affinity changes for various mismatched base-pairs.

Short antisense oligonucleotides alleviate the pleiotropic toxicity of RNA harboring expanded CGG repeats

Fragile X-associated tremor/ataxia syndrome (FXTAS) is an incurable neurodegenerative disorder caused by expansion of CGG repeats in the FMR1 5’UTR. The RNA containing expanded CGG repeats (rCGGexp) causes cell damage by interaction with complementary DNA, forming R-loop structures, sequestration of nuclear proteins involved in RNA metabolism and initiation of translation of polyglycine-containing protein (FMRpolyG), which forms nuclear insoluble inclusions. Here we show the therapeutic potential of short antisense oligonucleotide steric blockers (ASOs) targeting directly the rCGGexp. In nuclei of FXTAS cells ASOs affect R-loop formation and correct miRNA biogenesis and alternative splicing, indicating that nuclear proteins are released from toxic sequestration. In cytoplasm, ASOs significantly decrease the biosynthesis and accumulation of FMRpolyG. Delivery of ASO into a brain of FXTAS mouse model reduces formation of inclusions, improves motor behavior and corrects gene expression profile with marginal signs of toxicity after a few weeks from a treatment.

Sensing and Signaling of Methionine Metabolism

Availability of the amino acid methionine shows remarkable effects on the physiology of individual cells and whole organisms. For example, most cancer cells, but not normal cells, are hyper dependent on high flux through metabolic pathways connected to methionine, and diets restricted for methionine increase healthy lifespan in model organisms. Methionine’s impact on physiology goes beyond its role in initiation of translation and incorporation in proteins. Many of its metabolites have a major influence on cellular functions including epigenetic regulation, maintenance of redox balance, polyamine synthesis, and phospholipid homeostasis. As a central component of such essential pathways, cells require mechanisms to sense methionine availability. When methionine levels are low, cellular response programs induce transcriptional and signaling states to remodel metabolic programs and maintain methionine metabolism. In addition, an evolutionary conserved cell cycle arrest is induced to ensure cellular and genomic integrity during methionine starvation conditions. Methionine and its metabolites are critical for cell growth, proliferation, and development in all organisms. However, mechanisms of methionine perception are diverse. Here we review current knowledge about mechanisms of methionine sensing in yeast and mammalian cells, and will discuss the impact of methionine imbalance on cancer and aging.

The state of oligomerization of Rubisco controls the rate of LSU translation in Chlamydomonas reinhardtii

Ribulose 1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) is a key enzyme for photosynthesis-driven life on Earth. While present in all photosynthetic organisms, its most prominent form is a hetero-oligomer in which a Small Subunit (SSU) stabilizes the core of the enzyme built from Large Subunits (LSU), yielding, after a chaperone-assisted multistep assembly, a LSU8SSU8 hexadecameric holoenzyme. Here we use Chlamydomonas reinhardtii, and a combination of site-directed mutants, to dissect the multistep biogenesis pathway of Rubisco in vivo. We identify assembly intermediates, in two of which LSU is associated with the RAF1 chaperone. Using genetic and biochemical approaches we further unravel a major regulation process during Rubisco biogenesis which places translation of its large subunit under the control of its ability to assemble with the small subunit, by a mechanism of Control by Epistasy of Synthesis (CES). Altogether this leads us to propose a model where the last assembly intermediate, an octameric LSU8-RAF1 complex which delivers LSU to SSU to form the Rubisco enzyme, converts to a key regulator form able to exert a negative feed-back on the initiation of translation of LSU, when SSU is not available.