The Neurospora crassa arg-2 gene encodes the small subunit of carbamoyl phosphate synthetase, the first enzyme in fungal arginine (Arg) biosynthesis. The arginine attenuator peptide (AAP), specified by an upstream open reading frame (uORF), stalls ribosomes at its termination codon in response Arg to control the translation of arg-2. In project 1, the effect of AAP and Arg on ribosome peptidyl transferase center (PTC) activity was analyzed in N. crassa and wheat germ cell-free translation extracts using the transfer of nascent AAP to puromycin as an assay. The results show that inhibition of PTC activity by the AAP and Arg is the basis for the AAP’s function. The mode of PTC inhibition appears unusual because neither a specific amino acid nor a specific nascent peptide chain length was required for AAP to function. In eukaryotic translation initiation, the stringency of start codon selection impacts initiation efficiencies at AUG codons in different contexts and at near-cognate codons (NCCs) that differ from AUG by a single nucleotide. In project 2, a codon-optimized firefly luciferase reporter was used to examine the stringency of start codon selection in N. crassa. In vivo and in vitro results indicated that the hierarchy of initiation in N. crassa is similar to that in human cells. The preferred context was more important for efficient initiation from NCCs than from AUG. In project 3, the use of NCCs was also specifically examined for the N. crassa cpc-1 gene. cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying a transcription activator, which drives the primary transcriptional response to amino acid starvation. In vitro studies showed that uORF1 and uORF2 in cpc-1 are functionally analogous to uORF1 and uORF4 in GCN4. uORF1 promotes reinitiation at downstream start codons. uORF2 inhibits translation from the main cpc-1 start codon. Four NCCs in the CPC1 reading frame and upstream of uORF2 can also be used for translation initiation. In summary, we explored uORF-mediated translational regulation and the use of NCCs as initiation codons. Taken together, these studies establish N. crassa as a model system to examine mechanisms contributing to translational control including initiation and termination.

Apolipoprotein B (apoB) is a key structural and functional protein of lipoproteins and is synthesized constitutively in the liver. This study investigated the role of microRNAs (miRNAs) in translational control of apolipoprotein B (apoB) mRNA and protein synthesis. Using bioinformatic analysis, I identified two specific miRNAs namely, miR-544 and miR-1202 with potential to interact with 3’ and 5’ UTR of apoB, respectively. Using HepG2 cells as the model system, the effects of transfection of exogenous miRNAs and inhibition of endogenous miRNAs were assessed on the expression of apoB mRNA and protein synthesis, as well as apoB mRNA traffic into cytoplasmic P-bodies. miR-544 induced a significant reduction in apoB mRNA expression and protein synthesis while increasing the co-localization of apoB mRNA into P-bodies. In contrast, transfection of miR-1202 increased apoB mRNA expression and protein synthesis. In summary, these data demonstrate that specific miRNAs are involved in translational control of apoB mRNA.

Apolipoprotein B (apoB) is a key structural and functional protein of lipoproteins and is synthesized constitutively in the liver. This study investigated the role of microRNAs (miRNAs) in translational control of apolipoprotein B (apoB) mRNA and protein synthesis. Using bioinformatic analysis, I identified two specific miRNAs namely, miR-544 and miR-1202 with potential to interact with 3’ and 5’ UTR of apoB, respectively. Using HepG2 cells as the model system, the effects of transfection of exogenous miRNAs and inhibition of endogenous miRNAs were assessed on the expression of apoB mRNA and protein synthesis, as well as apoB mRNA traffic into cytoplasmic P-bodies. miR-544 induced a significant reduction in apoB mRNA expression and protein synthesis while increasing the co-localization of apoB mRNA into P-bodies. In contrast, transfection of miR-1202 increased apoB mRNA expression and protein synthesis. In summary, these data demonstrate that specific miRNAs are involved in translational control of apoB mRNA.

Inactive mutants of the ped gene cause two phenotypes in Drosophila melanogaster: male sterility and the early translation of DHODH within spermatogenesis. Investigation of the PED amino acid sequence revealed an OTU domain and an ubiquitin interacting motif, suggesting that it is a member of the otubain sub-family of de-ubiqutinating enzymes. To test this, the putative active cysteine residue was mutated. Results show that this single cysteine residue is required for ped to confer male fertility. Purified wild type PED was also used to carry out in vitro deubiquitinating assays. These assays failed to show any ability for PED to cut ubiquitin chains of varying length or linkage type. Previously, a translational control element was identified in dhod mRNA which is required for its early translation phenotype in ped mutants. In an attempt to identify additional transcripts that have their translational timing affected by PED, the don juan-like 5′ UTR was inserted into a reporter gene and examined in a ped mutant background. No delay of this reporter gene was observed suggesting that don juan-like mRNA is not under the exact control pathway that dhod is.

The study of proteins and their function is key to understanding the intrinsic properties of the cell both in normal and disease states. An important part of this analysis involves the prediction and elucidation of three-dimensional protein structures, and the interactions they undertake, through the use of computational techniques. The majority of work in this thesis focuses on the effects that non-specific charge interactions have on these structures. Firstly, sets of proteins that select a single partner from closely related alternatives were analysed using an empirical binding model in order to identify a determinant for binding specificity. Here, we predicted that charge interactions are more favourable in the majority of cognate pairs, compared to other energetic and geometric properties. In addition to this, charged protein side-chains were found to be important with respect to phosphorylation sites that lie in disordered regions of proteins. The analysis of charge environments around these sites indicated a propensity for a subset of residues to be phosphorylated when surrounded by charged residues. This was especially true for proteins involved in RNA processing.An investigation of protein-mRNA interactions also identified a role for charge interactions that occur within translational control mechanisms. The correlation seen between positively charged disordered regions of specific regulatory proteins and the secondary structure of target mRNAs revealed a potential control mechanism that is partially influenced by polyelectrostatic interactions.

Indiana University-Purdue University Indianapolis (IUPUI) / In response to environmental and physiological stresses, cells impose translational control to reprogram adaptive gene expression and conserve energy and nutrients. A central mechanism regulating translation involves phosphorylation of the a-subunit of the eukaryotic initiation factor -2 (p-eIF2a), which reduces delivery of initiator tRNA to ribosomes and represses global protein synthesis. The pathway featuring p-eIF2a is called the integrated stress response because it involves multiple related eIF2a kinases, each responding to different stress arrangements. While p-eIF2a limits global protein synthesis, a subset of mRNAs are preferentially translated in response to p-eIF2a. Preferential translation of stress adaptive mRNAs is regulated by upstream opening reading frames (uORFs) present in the 5’-leader of these transcripts. In most cases uORFs are inhibitory in nature, but in some case uORFs can instead promote the translation of the downstream CDS. This study is focused on preferential translation of the gene Inhibitor of Bruton’s Tyrosine Kinase-alpha (IBTKa) in response to endoplasmic reticulum stress. The human IBTKa gene encodes a 1353 amino acid residue protein, along with a 5’-leader featuring predicted canonical uORFs. Among the four predicted uORFs, the 5'-proximal uORF1 and uORF2 are phylogenetically conserved among mammals and are well translated as judged by reporter assays, whereas uORF3 and uORF4 are not conserved and are poorly translated. In addition to the uORFs in the IBTKa mRNA, a phylogenetically conserved stem-loop (SL) of moderate stability is present 11 nucleotides downstream of uORF2. Using luciferase reporter assay, the uORF2 and SL were shown to function together to repress the translation of human IBTKa. In non-stressed conditions, the SL combined with uORF2 are critical for reducing ribosomes from reinitiating at the IBTKa coding sequence (CDS), thus repressing IBTKa expression. Upon ER stress and induced p-eIF2a, the more modestly translated uORF1 facilitates the bypass of the inhibitory uORF2/SL to enhance the translation of main CDS of IBTKa. This study demonstrates that uORFs in conjunction with RNA secondary structures can be critical elements that serve as a “bar code” by which scanning ribosomes decide which mRNAs are preferentially translated in the integrated stress response. / 2023-10-03

Activity-dependent and synapse-specific translation of mRNAs is required for long-term changes in synaptic strength (or efficacy). However, many of the components mediating repression, transport and activation of mRNAs are unknown. Translational control in neurons is a highly conserved process and mediated by a ribonuclear particle (RNP). This study shows that RNPs in Drosophila neurons are similar not only to mammalian neuronal RNA granules but also to yeast P-bodies, cytoplasmic foci involved in translational repression and RNA decay. The evolutionarily conserved proteins Me31b and Trailer Hitch localize to RNA granules. Me31b and Trailer Hitch are required for normal dendritic growth. Mutations in Me31b and Trailer Hitch suppress phenotypes resulting from overexpression of Fragile X Mental Retardation protein, suggesting that both proteins may act as translational repressors. In addition, this study reports the identification of novel translational repressors in neurons. Using the overexpression phenotype of Fragile X Mental Retardation protein in a candidate-based genetic screen, I identified dominant suppressor mutations in five genes, including Doubletime/Discs Overgrown, Orb2/CPEB, PolyA Binding Protein, Rm62/Dmp68 and SmD3. Like Me31b and Trailer Hitch, all five proteins localize to neuronal RNPs. Overexpression of each proteins affects dendritic branching of sensory neurons in Drosophila. Identification and further characterization of these novel RNP granule components and dFMR1-interacting proteins may provide further insights into the mechanisms controlling translational in dendrites.

The subcellular localisation of mRNA is a widespread mechanism to determine the fate of mRNAs in eukaryotes. Translationally repressed mRNAs localise to P-bodies and stress granules where their decay and storage, respectively, are directed. In a study from the Ashe lab, specific mRNAs were identified to localise, in actively growing S. cerevisiae, to cytoplasmic granules that do not seem to be related to P-bodies or stress granules but appear to be associated with active translation (Lui et al., 2014).It is possible that this might represent a strategy to co-regulate the expression of proteins from the same pathway. In the work of this thesis, microscopy techniques to visualise RNAs in live cells were used to extend the localisation analysis to several mRNAs encoding translation factors. The investigated transcripts were all found to localise to mostly one or two cytoplasmic granules per cell and would sometimes overlap with other transcripts, suggesting that each granule contains a mixture of mRNAs. Granules tend to migrate to the bud tip and may provide the daughter cell with a "start-up kit" of transcripts essential for rapid growth. A similar pattern can be observed in yeast cells growing undergoing filamentous growth, with granules harbouring translation factor transcripts often found in the apical quarter of the elongated cell. Although the mechanism by which the granules form and their protein composition are not yet known, high-throughput genetic screens performed as part of this work offer some insight into factors that might be involved in granule assembly and proteins that partially overlap with the granules. We propose that granules containing translation factor mRNAs might be functioning as a specialised factory for the translation machinery and are possibly being directed to the point in the cell where the rhythm of protein production is highest.

Establishment of cell polarity requires the involvement of several posttranscriptional regulatory mechanisms, including mRNA localization and translational control. A family of highly conserved RNA binding proteins in vertebrates, VICKZ (V̲g1RBP/V̲era, I̲MP-1, 2, 3, C̲RD-BP, K̲OC, Z̲BP-1) proteins, has been shown to act in these two processes. Previous studies of the posttranscriptional mechanisms mediated by VICKZ family members have been largely limited by the lack of genetic approaches in certain vertebrate systems. Identification of Imp, the Drosophila member of the VICKZ family, opened the possibility to use genetic approaches to investigate the roles of a VICKZ family member in mRNA localization and translational control. In this dissertation, we show that Imp is associated with Squid and Hrp48, two heterogeneous proteins (hnRNP) that complex with one another to regulate localized expression of gurken (grk). In addition, Imp binds grk mRNA with high affinity in vitro and is concentrated at the site of grk localization in midstage oocytes. Mutation of the Imp gene does not substantially alter grk expression, but does partially suppress the grk mis-expression phenotype of fs(1)k10 mutants. In contrast, overexpression of Imp in germ line cells results in mislocalization of grk mRNA and protein. The opposing effects of reduced and elevated Imp activities on grk expression suggest that Imp acts in regulation of grk expression, but in a redundant way. To further explore the mechanisms by which localized expression of grk is regulated by Imp, a deficiency screen was conducted to search for dominant modifiers of the dorsalized phenotype resulting from Imp overexpression. Twelve genomic regions were identified to contain dominant modifiers of the Imp overexpression phenotype. Further characterization of mutants of genes within these genomic regions led to identification of five modifiers, including cyclin E (cycE), E2f transcriptional factor 1 (E2f1), lingerer (lig), snail (sna) and mushroom body expressed (mub). E2f1 encodes a transcriptional factor that is involved in regulating the G1 to S phase transition during mitosis. Mutation of E2f1 results in altered grk mRNA and protein distribution within oocyte, revealing a role for this gene in regulation of grk expression. / text

Il a récemment été rapporté que l'interaction physiologique entre la protéine nucléaire SMRT et le facteur de transcription cellulaire STAT3 pouvait être rompue par la protéine EBNA2 du virus Epstein-Barr (EBV). La liaison de SMRT à STAT3 diminue son activité de transcription. EBNA2 libère SMRT de ce complexe augmentant ainsi la transcription des gènes régulés par STAT3. STAT3 régule de nombreux effets immunodépresseurs mais aussi anti-prolifératifs et anti-apoptotiques dans l'organisme hôte, et pourrait jouer un rôle important dans la stratégie de survie du virus Epstein-Barr. EBNA2 et STAT3 sont tous deux intrinsèquement désordonnés (IDPs) comme un tiers de toutes les protéines eucaryotes et 70% des protéines associées aux cancers. Toutefois, malgré leur abondance, notre compréhension de leurs fonctions reste limitée notamment à cause de la difficulté à les produire en quantités suffisantes pour réaliser des études structurales et biophysiques. Afin de surmonter cette difficulté, la technologie ESPRIT a été utilisée pour générer des fragments de SMRT et EBNA2 solubles et stables. Ces fragments se répartissaient sur une grande partie des gènes d'EBNA2 et SMRT pour lesquels les protéines exprimées étaient de tailles compatibles avec des expériences de RMN. Ceci a permis de réaliser des études d'interaction avec STAT3. Les deux protéines étant majoritairement intrinsèquement désordonnées, toutes les données d'interaction ont été analysées pour EBNA2 et SMRT avec une attention particulière sur les propriétés de protéines désordonnées. Des fragments des deux protéines, EBNA2 et SMRT, ont été soumis à des tests d'interaction avec le dimère de STAT3 en utilisant des méthodes biophysiques variées. Ainsi la cinétique de liaison et la séquence d'acides aminés impliqués dans l'interaction ont pu être déterminées. Etant donné son importance biologique et son interaction de haute affinité, l'accent a été porté sur l'interaction EBNA2-SMRT. Ainsi des résultats préliminaires provenant d'investigations en cellules de mammifères ont pu être obtenus. En outre l'interaction entre EBNA2 et d'autres protéines cellulaires a été brièvement étudiée. Les résultats obtenus ont pour objectif d'améliorer notre compréhension de la stratégie avec laquelle EBV parvient, avec succès, à persister toute une vie dans l'organisme hôte. / It was recently reported that the physiological interaction between the nuclear protein SMRT and the cellular transcription factor STAT3 could be disrupted by the Epstein-Barr-virus protein EBNA2. Binding of SMRT to STAT3 decreases its transcriptional activity. EBNA2 releases SMRT from this complex and therefore enhances the transcription of both host and viral STAT3 regulated genes. STAT3 regulates several immunosuppressive as well as pro-proliferative and anti-apoptotic effects in the host organism and so may have importance for the viral survival strategy of Epstein-Barr-virus. Both EBNA2 and SMRT are intrinsically disordered proteins (IDPs) in common with about one third of all eukaryotic proteins and 70% of cancer-related. Despite their abundance, our understanding of how they function is limited, in part due to difficulties in production of stable samples in the large quantities necessary for structural and biophysical studies. The EPRIT technology was used to overcome these problems and generate well-behaving and -expressing EBNA2 and SMRT fragments. These fragments covered large parts of the EBNA2 and SMRT genes and were in a suitable size for NMR experiments. This enabled interaction studies of these proteins with STAT3. As both proteins are mainly intrinsically disordered all interaction data were analysed with attention to the disordered properties of both proteins. In order to complement the interaction data all fragments used for the interaction studies were as well characterized as IDPs using biochemical and biophysical methods. Fragments of both proteins, EBNA2 and SMRT together with the STAT3 dimer were subjected to binding analysis using various biophysical methods. The kinetics of the binding could be determined and the binding regions could be narrowed down to the amino acid level. Because of its biological significance and higher affinity interaction the EBNA2-STAT3 part was prioritized and first preliminary results investigating the interaction in mammalian cells could be obtained. Also, the interaction between EBNA2 and other cellular proteins was studied briefly. The obtained results aim to improve our understanding of how EBV succeeds to persist lifelong in the host organism.