Eukaryotic translation initiation factor 2 (eIF2) is a heterotrimeric G-protein that plays a critical role in protein synthesis regulation. eIF2-GTP binds Met-tRNAi to form the eIF2-GTP:Met-tRNAi ternary complex (TC), that is recruited to the 40S ribosomal subunit. Following GTP hydrolysis, eIF2-GDP is recycled back to TC by its guanine nucleotide exchange factor (GEF), eIF2B. Phosphorylation of the eIF2α subunit in response to various cellular stresses converts eIF2 into a competitive inhibitor of eIF2B, triggering the integrated stress response. Dysregulation of eIF2B activity is associated with a number of pathologies, including neurodegenerative diseases, metabolic disorders, and cancer. However, despite decades of research, the underlying molecular mechanisms remain unknown. This is due in large part to the absence of a structural understanding of the eIF2B assembly and of the eIF2B:eIF2 interaction. Common methods, such as yeast genetics, have been unable to unambiguously determine these mechanisms. Meanwhile, expanded interest in the integrated stress response has uncovered a diverse array of pathologies for which therapeutic modulation of the eIF2B:eIF2 interaction may ameliorate or overcome disease states. In this dissertation, a combination of structural and biochemical techniques is employed to elucidate the mechanisms of eIF2B action and its regulation by eIF2α phosphorylation. The aim is to provide a direct, unambiguous, structural understanding of eIF2B assembly and of eIF2B’s interactions with phosphorylated and unphosphorylated eIF2α. The work described here was among the first to challenge the widely held notion of a pentameric eIF2B assembly, as eventually confirmed by the recent publication of eIF2B’s crystal structure. The work further aims to overturn another long-standing assumption regarding the nature of inhibition of eIF2B activity: that competitive inhibition is mediated by a “direct effect” of the negatively charged phosphate group on the eIF2α:eIF2B interaction. Instead, we present evidence for an “indirect effect,” whereby phosphorylation disrupts a novel intramolecular interface within eIF2α, exposing an eIF2α surface that binds eIF2B and is responsible for inhibition of eIF2B. In the end, we combine a structural model of the eIF2B:eIF2 complex with our novel mechanism of inhibition, placing them within the larger thermodynamic context of eIF2-GDP recycling by eIF2B. / 2017-09-08T00:00:00Z

No description available.

Gap junctions, comprised of connexin proteins, are essential for direct intercellular electrical, metabolic, and immunological coupling. Connexin43 (Cx43, gene name GJA1) is the most ubiquitously expressed gap junction protein, and Cx43 gap junctions are altered in pathological states including cardiac disease and cancer. The GJA1 mRNA undergoes alternative translation initiation to yield a truncated Cx43 isoform, GJA1-20k, that can regulate gap junction formation. Using epithelial-mesenchymal transition (EMT) as a cellular model of gap junction remodeling, we have demonstrated altered translation initiation of Gja1 as a mechanism by which cellular Cx43 gap junctions can be dynamically regulated. Suppression of Gja1 alternative translation is necessary for Cx43 gap junction loss, and stable expression of GJA1-20k rescues gap junction formation during EMT. To identify regulatory factors acting on the Gja1 mRNA, an MS2 RNA aptamer tagging system was adapted to isolate Gja1 with associated RNA binding proteins. We find the RNA binding protein IMP1 is sensitive to hypoxic stress and complexes with Gja1 mRNA, where it is necessary for alternative translation to generate GJA1-20k. We have demonstrated alterations in translation initiation of the Gja1 mRNA as a critical mechanism by which cells modulate Cx43 gap junctional coupling in changing conditions and identified a novel regulator of this process in mammalian cells. / Doctor of Philosophy / Communication between cells is necessary for healthy function of organs throughout the body. Gap junctions form conduits through which signals can pass directly between neighboring cells. Many diseases, including cancer and heart disease, involve disturbances in gap junction communication. Connexin proteins are the building blocks of gap junctions, and it was recently demonstrated that smaller fragments of connexins are synthesized by cells by a poorly understood process called alternative translation. Importantly, levels of these connexins fragments can alter gap junction formation. We have used mammalian cells to delineate the mechanism by which this alternative protein translation regulates gap junction formation and generated insight into how such protein synthesis is dynamically regulated. Harnessing this knowledge will inform development of new therapeutics inducing alternative translation to rescue gap junctions, and restore normal communication in pathological conditions.

No description available.

The studies reported in this thesis address firstly, the biology of a modified nucleoside, Queuosine (Q) and secondly, the properties of mitochondrial translation initiation factor 2. A summary of the relevant literature on both these topics is presented in Chapter 1. Section I of this ‘General Introduction’ summarizes the literature on biosynthesis and physiological importance of Queuosine. Section II is a brief review of the current understanding of translation initiation in Eubacteria. Information about the mitochondrial translation initiation apparatus also features as a subsection. The next chapter (Chapter 2), describes the ‘Materials and Methods’ used throughout the experimental work presented in the thesis. It is followed by three chapters containing experimental work as described below:- i) Biosynthesis of Queuosine (Q) in Escherichia coli Q is a hypermodification of guanosine found at the wobble position of tRNAs with GUN anticodons. Q is thought to be produced via a complex multistep pathway, the details of which are not known. It was found in our laboratory that a naturally occurring strain of E. coli B105 lacked Q modification in the tRNAs. As the known enzymes of Q biosynthesis were functional in this strain, it presented us with the opportunity to uncover novel component(s) of Q biosynthetic pathway. In the present work, a genetic screen was developed to map the defect in E. coli B105 to a previously uncharacterised gene, ybaX, predicted to code for a 231 amino acid long protein with a pI of 5.6. Further genetic analyses showed that YbaX functions at a step leading to production of preQ0, the first known intermediate in the generally accepted pathway that utilizes GTP as the starting molecule. The gene ybaX has been renamed as queC. Using a combination of bioinformatics based prediction and gene knockouts, we have also been able to place two more genes, queD and queE at the initial step in Q biosynthesis, suggesting that the initial reaction of Q biosynthesis might be more complex and mechanistically different than what has been proposed earlier. ii) Caenorhabditis elegans as a Model System to Study Queuosine Metabolism in Metazoa Animals are thought to obtain Q (or its analogs) as a micronutrient from dietary sources such as gut microflora, and the corresponding base is then inserted in the substrate tRNAs by tRNA guanine transglycosylase (TGT). In animal cells, changes in the abundance of Q have been shown to correlate with diverse phenomena including stress tolerance, cell proliferation and tumor growth but the precise function of Q in animal tRNAs remains unknown. A major obstacle in the study of Q metabolism in higher organisms has been the requirement of a chemically defined medium to cause Q depletion in animals. Having discovered that E. coli B105 has a block in the initial step of Q biosynthesis, we reasoned that this strain could be used as a Q- diet for organisms like C. elegans, which naturally feed on bacteria. An analysis of C. elegans tRNA revealed that as in the other higher animals, tRNAs in the worm C. elegans, are modified by Q and its sugar derivatives. When the worms were fed on Q deficient E. coli B105, Q modification was absent from the worm tRNAs suggesting that C. elegans lacks a de novo pathway of Q biosynthesis. The inherent advantages of C. elegans as a model organism, the speed and simplicity of conferring a Q deficient phenotype on it, make it an ideal system to investigate the function of Q modification in tRNA. By microinjecting tgt-1-gfp constructs into C. elegans, we could also demonstrate that a major form of TGT is localised to the nucleus, suggesting that insertion of Q into the tRNAs could be occurring in the nucleus. iii) Dual Function of Bovine Mitochondrial Initiation Factor 2 as Initiation Factors 1 and 2 in Escherichia coli Translation initiation factors 1 and 2 (IF1 and IF2) are known as ‘universal translation initiation factors’ due to the presence of their homologs in all living organisms. Homologs of these factors are also present in the chloroplast, however, a unique situation exists in the mitochondria where IF2 homolog (IF2mt) is known to occur but an IF1 like factor is not found. We have engineered a system of E. coli knockouts to allow the study of IF2mt in a prokaryotic milieu. We found that the bovine IF2mt complements an E. coli strain wherein the gene for IF2 is knocked out, providing the first proof of a mitochondrial translation initiation factor working in a eubacterial system. This conservation of function is especially interesting in light of the recent reports revealing significant differences between the mitochondrial and eubacterial ribosomes. Further, we found that the IF2mt can also support a double knockout of IF1 and IF2 genes in E. coli, suggesting that IF2mt possesses both IF1 and IF2 like activities in E. coli. This finding offers an explanation for the lack of an IF1 like factor in mitochondria. Molecular modeling of bovine IF2mt indicated that a conserved insertion found in all mitochondrial IF2s, may form a protruding α-helix that could stabilize IF2mt on ribosomes. This insertion could in principle function as IF1 and we have explored the role of this conserved insertion both in vivo and in vitro, by generating mutants of IF2mt and EcoIF2, to lose or gain the conserved insertion respectively.

The DEAD-Box RNA helicase family is a conserved group of enzymes that function in gene expression through ATP-dependent RNA unwinding and ribonucleoprotein (RNP) remodeling. DEAD-Box helicases function in multiple cellular processes, including pre-mRNA processing, translation, mRNA export, and mRNA decay. Although DEAD-Box proteins are critical for gene expression, much of their mechanistic activities are poorly understood. DEAD-Box proteins have increasingly been linked to tumorigenesis in humans, and better defining their activity at the mechanistic level will aid in understanding the underlying disease pathology. In this study, we used the model organism Saccharomyces cerevisiae to study the human DEAD-Box protein DDX3 orthologue, Ded1, and its role in translation initiation during cellular stress. Recently, we have found that Ded1 is an important mediator of the cellular stress response in a TOR-dependent manner. TOR regulates protein synthesis dependent on energy availability in the cell by regulating the assembly of the eukaryotic translation initiation complex. Human DDX3 has been found to interact with translation initiation complex subunit eIF4E and Ded1 has been found to interact with the translation initiation complex subunit eIF4G. In this study, we examined the purported interaction region between Ded1 and eIF4G on the C-terminus of Ded1 and found that ded1 Δ591-604 prevents eIF4G degradation under rapamycin treatment and confers resistance to rapamycin-induced growth inhibition. We also examined putative regulatory phosphorylation sites in the purported Ded1 eIF4G binding region. We propose that the Ded1/eIF4G interaction is critical for the repression of translation by Ded1 and that eIF4G degradation may be regulated by Ded1 under stress conditions.

Protein kinases ERK1 and ERK2 are one of the most studied proteins in cell signalling. Both proteins are involved in a plethora of processes, such as phosphorylation and activation of kinases as part of signalling pathways. Enzymes ERK1 and ERK2 are part of MAPK/ERK signalling cascade, connected to many cellular including cell proliferation, cell growth or differentiation. The MAPK/ERK signalling cascade is often activated in different types of tumors, making it a candidate for developing new chemical inhibitors. One of the important questions in fundamental research of ERK1 and ERK2 protein kinases is the search for difference between these proteins. Current knowledge points to redundancy of both proteins, howver several examples suggest otherwise. Recently, the work presented in Casanova et al. 2012 indirectly suggests divergent effect of ERK1 and ERK2 on cap-independent translation initiation. In the Laboratory of RNA biochemistry we focus on HCV IRES (Hepatitis C Virus Internal Ribosome Entry Site) dependent translation initiation. This diploma thesis lead to establish RNA interference method in our laboratory and to establish reporter system to study ERK1 and ERK2 effect on HCV IRES dependent translation initiation. Based on our data acquired during our research, we present in this work...

In eukaryotes, translation initiation is guided by up to twelve protein initiation factors (eIFs) and begins with the formation of the 43S pre-initiation complex (PIC) composed of the small ribosomal subunit (40S), eIF2.GTP/Met-tRNAi Met ternary complex, and eIFs 1, 1A, 3 and 5. The 43S PIC subsequently interacts with the 5'end of an mRNA (an mRNA recruitment step) and thus formed 48S PIC travels in 5' to 3' direction along the mRNA leader sequence to locate the AUG start codon (this presumably linear movement is generally known as scanning). Start site selection results in the dissociation of the initiation factors and joining of the large (60S) ribosomal subunit to form the 80S initiation complex poised for elongation. Eukaryotic initiation factor 3 (eIF3) plays a critical role in most of these events; however, the molecular details of most of its contributions are still unknown to us. Previous in vivo studies generated numerous mutations in all eIF3 subunits with specific defects either in the PICs assembly or in the following steps such as scanning, AUG recognition, etc. To understand the exact role of eIF3 in this intriguing process at the molecular level, we have embarked on a study that aims to dissect the individual functions of each eIF3 subunit in translation initiation using the purified...

L’initiation de la traduction est un processus complexe qui fait intervenir une douzaine de facteurs d’initiation. L’élément clef de ce mécanisme est le facteur eIF4E qui grâce à sa liaison avec la coiffe, recrute l’ensemble du complexe d’initiation au niveau de l’ARNm et permet l’assemblage du ribosome au voisinage du codon d’initiation. Chez Arabidopsis thaliana, il existe à coté de la protéine eIF4E, une isoforme : eIF(iso)4E. Ces deux protéines participent à l’initiation de la traduction. L’existence de ces deux protéines évoque un phénomène de redondance fonctionnelle qui est attestée par la létalité du double mutant alors que les simples mutants sont viables. Cependant, l’étude phénotypique de mutants pour les gènes eIF4E et eIF(iso)4E a permis de montrer que cette redondance est partielle et inégale. En effet, les mutants pour le gène eIF4E présentent un retard de croissance, un retard de floraison, une baisse de la fertilité, une sénescence précoce et une activité traductionnelle réduite. Inversement, le phénotype des plantes mutantes pour le gène eIF(iso)4E est comparable à celui du sauvage. Les mutations dans les gènes eIF4E et eIF(iso)4E induisent une hypersensibilité à la lumière Enfin, en présence d’un inhibiteur de TOR la croissance de la racine des plantes de la lignée mutante pour le gène eIF(iso)4E est moins inhibée que celle des plantes de la lignée sauvage. / More than 12 initiation factors are involved in eukaryotic translation initiation. The key step of this mechanism is the binding of eIF4E with the cap of the mRNA. This step allows the recruitment of the initiation complex and the assembly of the ribosome close to the start codon. Arabidopsis thaliana encodes a second eIF4E protein: eIF(iso)4E. Those two proteins perform translation initiation. The existence of those two proteins suggests that they may be functionally redundant. Double mutant lethality testifies for functional redundancy. However, phenotypic studies of mutant lines for gene eIF4E and eIF(iso)4E showed that redundancy is partial and unequal. Indeed, the eIF4E mutant lines exhibit growth delay in rosette and roots, bolting delay, impaired fertility and early senescence in leaves. Translational activity is also largely impaired. On the contrary, a mutant line for the eIF(iso)4E gene has the same phenotype as wild type line. Mutant lines for eIF4E and eIF(iso)4E are more sensitive to light and accumulate anthocyanins even in normal light. On the molecular level, the amounts of mRNA of genes that are involved in high light response and their association to polysomes increase. When plants are grown on media containing a TOR inhibitor, AZD-8055, plants of the eIF(iso)4E mutant line show less root growth inhibition compared to wild type and eIF4E mutant lines. This result suggests that eIF(iso)4E could be targeted by the TOR pathway.

Translation initiation is a multi-step process culminating in formation of the elongation- competent 80S ribosome. It requires accurate assembly of small and large ribosomal subunits, mRNA, initiation Met-tRNAi Met and at least 12 eukaryotic initiation factors (eIFs). This phase of protein synthesis is also one of the key points of regulation of gene expression. One of the main aims of our laboratory is a complex characterization of the multiprotein eIF3 complex that has been implicated in most of the steps of translation initiation. For example, we revealed and described its novel role in translation reinitiation (REI), a gene-specific translational control mechanism that among others governs expression of an important yeast transcriptional activator GCN4. Here I present a detailed characterization of the multi-functional N-terminal domain of Tif32 (subunit eIF3a). We demonstrated that the Tif32-NTD functionally interacts with the 5' sequences of short upstream ORF (uORF1) in the GCN4 mRNA leader and thus allows efficient reinitiation downstream of this critical reinitiation-permissive uORF. Four REI- promoting elements (RPEs) were identified in the 5' sequences of uORF1, two of which were shown to work in the Tif32-NTD-dependent manner. The structure of the 5' sequences was determined...