Thesis (Ph.D.)--Ohio University, March, 2009. / Release of full electronic text on OhioLINK has been delayed until April 1, 2010. Includes bibliographical references (leaves 70-91)

Thesis (Ph.D.)--Ohio University, March, 2009. / Title from PDF t.p. Release of full electronic text on OhioLINK has been delayed until April 1, 2010. Includes bibliographical references (leaves 70-91)

Thesis (Ph. D.)--Ohio State University, 2008. / Title from first page of PDF file. Includes vita. Includes bibliographical references (p. 119-137).

Thesis (Ph. D.)--Miami University, Dept. of Microbiology, 2004. / Title from second page of PDF document. Includes bibliographical references (p. 139-145).

Termination of translation in eukaryotes occurs when a translating ribosome encounters a stop codon (UAA, UAG, or UGA) in its A site. This triggers the recruitment of translation termination factors eRF1, a tRNA-mimicking protein responsible for decoding the stop codon and catalyzing peptide release, and eRF3, a translational GTPase that stimulates peptide release in a GTP-hydrolysis-dependent manner. Upon successful stop codon decoding by eRF1, eRF3 carries out GTP hydrolysis and dissociates from the ribosome. eRF1 subsequently gets accommodated into the peptidyl transferase center (PTC) and catalyzes the release of the nascent peptide. The structures for the pre-accommodated eRF1 with eRF3 trapped on ribosome using non-hydrolysable GTP analogs as well as for the PTC-accommodated eRF1 have been solved using cryogenic electron-microscopy (cryo-EM). The structures reveal the binding mode and interactions between the release factors and the pre-termination complex. However, the mechanism of eRF3 GTPase activation and subsequent eRF1 accommodation into the PTC remains poorly understood. To address this knowledge gap, we used single-particle time-resolved cryo-EM (TRCEM) to capture the structures of intermediates formed during the termination process. For our TRCEM experiments, we first developed a Polydimethylsiloxane (PDMS)-based modular microfluidic mixing-spraying device with a SiO₂ internal coating. The device has a SiO₂-coated PDMS-based 3D splitting-and-recombination (SAR) micro-mixer capable of mixing two fluids within 0.5 ms with more than 90% efficiency. The SiO₂coating strengthens the PDMS channels and acts as a hydrophilic barrier preventing sample adsorption to the PDMS surface. The micro-mixer is connected to a glass microcapillary that acts as the reaction channel. Channels of different lengths can be used to vary the overall reaction time between 10 ms and 1000 ms. The microcapillary is connected to a 3D micro-sprayer for generating a 3D plume of sprayed droplets. A cryo-EM grid is passed through the spray cone to collect droplets and is rapidly plunged into liquid cryogen for vitrification. By using TRCEM as well as the conventional blotting method for cryo-EM sample preparation, we captured the reaction between a pre-termination (pre-TC) mimic and the ternary complex of eRF1, eRF3, and GTP at reaction times of 450 ms, 900 ms, 15 s, and 10 min. Classification of the cryo-EM data resulted in maps for five distinct factor-bound classes. Four maps belonged to intermediates with densities for eRF1 and eRF3 bound to the pre-TC in varying conformations. The fifth map had a density matching the PTC-accommodated eRF1. Population analysis allowed us to arrange the classes chronologically and track the events leading to GTPase activation during the termination process. Atomic model building and refinement allowed us to determine the hydrolysis state of the eRF3-bound GTP and revealed the catalytic mechanism for GTP-hydrolysis. The models revealed a potential mechanism for GDP dissociation post-GTP-hydrolysis.

The mRNA coding for the ammonium inducible NADP-specific glutamate dehydrogenase (NADP-GDH) from Chlorella was studied in induced and uninduced cells to determine the molecular mechanisms which regulate the cellular levels of this enzyme. A procedure for isolation of a high yield of total undegraded cellular polysomes was developed. The crosslinking reagent, dimethyl suberimidate, was employed to prepare a stable NADP-GDH-crosslinked-Sepharose-4B antigen affinity column for the purification of rabbit anti-NADP-GDH IgG. Binding studies with ¹²⁵I-labelled antibody and total polysomes, isolated from induced and uninduced cells, showed that the NADP-GDH was being synthesized on polysomes from both types of cells. When poly(A)- containing RNA was extracted from polysomes isolated from induced and uninduced cells, and translated in an mRNA-dependent in vitro translation system, NADP-GDH antigen was synthesized from the RNA from both sources. Based on sucrose density gradient analysis, Chlorella NADP-GDH mRNA has a sedimentation coefficient of 18 Comparison of the amounts of NADP-GDH synthesized in vitro from poly(A)-containing RNA and non-poly(A)-containing RNA showed the NADP-GDH mRNA contained polyadenylic acid sequence. By use of an indirect immunoadsorption procedure, the NADP-GDH mRNA was purified five- to sevenfold from total poly(A)-containing RNA. The overall purification of the NADP-GDH mRNA from total polysomal RNA was approximately two hundred-fold. Complementary DNA was synthesized from the partially purified RNA with reverse transcriptase. The cDNA sequences hybridized to the least abundant class of mRNA sequences present in total poly(A)-containing RNA. In vitro translation of total poly(A)-containing RNA showed that NADP-GDH synthesis was 0.1% of total protein synthesis. Upon addition of inducer to previously uninduced, synchronous cells, the amount of translatable NADP-GDH mRNA increased in a linear fashion after 30 min of the induction period. A change in rate of NADP-GDH mRNA accumulation was observed after 30 min of the induction period. The results support the prediction that since the NADP-GDH enzyme is unstable in vivo, during periods of NADP-GDH accumulation, the NADP-GDH mRNA accumulates. When poly(A)-containing RNA, isolated from uninduced synchronous cells was translated in vitro, NADP-GDH antigen was synthesized at each time in the cell cycle examined. The amount of translatable NADP-GDH mRNA increased throughout the cell cycle with a rate change occuring during the S-phase. This pattern of NADP-GDH mRNA accumulation is consistent with the hypothesis that NADP-GDH mRNA accumulates in uninduced cells at a rate proportional to gene dosage. These results provide one explanation for the observed pattern of enzyme potential in synchronous cells cultured in the absence of inducer. The data are consistent with the possibility that a single mRNA, which is subject to post-transcriptional modification by the inducer, codes for NADP-GDH. / Ph. D.

Calcium (Ca2+) plays an important role as a second messenger, transmitting the message of arrival of stimuli such as hormones and neurotransmitters to the intracellular system that carries out the cellular response to the stimulus. The universality of Ca2+ as an intracellular messenger depends on its enormous versatility. This versatility is exploited to control processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion, and must be accomplished within the context of Ca2+ being highly toxic. Ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs) are Ca2+ -release channels located on intracellular membranes of the endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) that perform essential functions as key targets of hormone/neurotransmitter action to initiate intracellular Ca2+ signals. The purpose of this project was to study the role of RyR2 in Ca2+ signaling in the NG115-401L neuronal cell line. siRNA transfection methods were employed to knockdown RyR2 expression levels in NG115-401L cells. We used reverse transcription and real-time PCR to evaluate RyR2 gene expression in transfected/untransfected cells. We also evaluated cytosolic Ca2+ changes induced by RyR activators or regulators, using fura-2 AM as the Ca2+ indicator. Successful RyR2 gene knockdown allowed us to carry out some initial experiments to characterize the specific roles played by the RyR2 receptor isoform. We examined cell responses to FK-506 under the condition of RyR2 knockdown, finding that RyR2 appears to confer selectivity to this response. Finally, the effects of siRNA transfection and FK-506 treatment on NG115-401L cell growth were evaluated. These experimental results may contribute to future studies of RyR2, and help develop novel treatments for RyR2-base d dysfunctional diseases.

Indiana University-Purdue University Indianapolis (IUPUI) / Gene expression is a highly coordinated process that relies upon appropriate regulation of translation for protein homeostasis. Regulation of protein synthesis largely occurs at the initiation step in which the translational start site is selected by ribosomes and associated initiating factors. In addition to the coding sequences (CDS) for protein products, short upstream open reading frames (uORFs) located in the 5’-leader of mRNAs are selected for translation initiation. While uORFs are largely considered to be inhibitory to translation at the downstream CDS, uORFs can also promote initiation of CDS translation in response to environmental stresses. Multiple transcripts associated with stress adaptation are preferentially translated through uORF-mediated mechanisms during activation of the Integrated Stress Response (ISR). In the ISR, phosphorylation of α subunit of the translation initiation factor eIF2α (eIF2α~P) during environmental stresses results in a global reduction in protein synthesis that functions to conserve energy and nutrient resources and facilitate reprogramming of gene expression. Many key regulators of the ISR network are subject to preferential translation in the response to eIF2α-P. These preferentially translated genes include the pro-apoptotic transcriptional activator Chop that modifies gene expression programs, feedback regulator Gadd34 that targets the catalytic subunit of protein phosphatase 1 to dephosphorylate eIF2α~P, and glutamyl-prolyl tRNA synthetase Eprs that increases the charged tRNA pool and primes the cell for resumption of protein synthesis after stress remediation. Ribosome bypass of at least one inhibitory uORF is a common theme between Chop, Gadd34, and Eprs, which allows for their regulated expression in response to cellular stress. However, different features encoded within the uORFs of the Chop, Gadd34, and Eprs mRNAs provide for regulation of their inhibitory functions, illustrating the complexities of uORF-mediated regulation of gene-specific translation. Importantly, preferentially translated ISR targets can also be transcriptionally regulated in response to cellular stress and misregulation of transcriptional or translational expression of Gadd34 can elicit maladaptive cell responses that contribute to disease. These mechanisms of translation control are conserved throughout species, emphasizing the importance of translation control in appropriate gene expression and the maintenance of protein homeostasis and health in diverse cellular conditions.

Originally 2A was characterised in foot-and-mouth disease virus. Site directed mutagenesis identified a C-terminus consensus motif [D(V/I)ExNPGP] and it is proposed that 2A interacts with the exit tunnel of the ribosome in a way that a specific peptide bond is skipped between the last glycine of 2A and the proline of 2B, thus providing a discontinuity in translation, resulting in release of discrete proteins from one single ORF. 2A was also identified in other picornaviruses, positive, single and double-stranded RNA insect viruses and mammalian rotaviruses. A motif present at the C-terminus of the 2A oligopeptide [D(V/I)ExNPGP] is very highly, though not completely conserved . The sequence upstream of this motif shows, however, no apparent conservation between 2As of different viruses. In this study, extensive site-directed mutagenesis were performed on several 2A sequences and a series of ‘hybrid' 2As comprising different consensus motifs juxtaposed with different upstream contexts were created as part of a detailed analysis of the mechanism of 2A-mediated ribosome stalling. The results demonstrated that a minimal region of twenty to twenty-three amino acids interacts with the exit tunnel of the ribosome to bring about a pause in processivity, alter the peptidyl transferase centre geometry and restrict the ribosome A site via two distinctive stalling mechanisms. Other molecular analyses tested here will require further optimisations or alternative methods: a visual method to explore the dynamics of re-initiation of translation from proline codon, purification of the translation-regulating factors and structural resolution of 2A sequences. Previously, cellular 2As were identified in non-LTR retrotransposons of trypanosomes. It is reported here as part of two other cellular organisms Saccoglossus kowalevskii (acorn worm) and Branchiostoma floridae (amphioxus). In the acorn worm, the nucleotides sequences corresponding to 2A motifs were part of the untranslated genome. In amphioxus, three 2A elements were identified in hypothetical proteins, and at the N-terminus of twenty non-LTR retrotransposons.

La protéine CIRP (Cold Induced RNA binding Protein) est une petite protéine de liaison à l’ARN de 172 acides aminés, qui est constituée du côté amino-terminal d’un domaine de liaison à l’ARN de type RRM (RNA recognition motif), et d’une partie carboxy-terminale riche en glycine et arginine qui comprend plusieurs motifs RGG. Elle a été identifiée comme étant inductible par hypothermie mais aussi par irradiation aux UV et par hypoxie. Nous avons analysé son expression et sa localisation en réponse à différents stress cellulaires. Nous avons montré qu’un traitement à l’arsénite qui induit un stress oxydant n’altère pas l’expression de CIRP provoque sa localisation dans les granules de stress (SG). Les SG sont des structures ribonucléoprotéiques cytoplasmiques contenant des complexes de pré-initiation incompétents pour la traduction, et qui s’accumulent dans les cellules exposées à un stress. Ces structures constituent des sites de triages des ARNm, dans lesquels les ARNm sont soit stockés en attente d’une réinitiation de la traduction une fois le stress surmonté, soit destinés à être dégradés. La protéine CIRP se localise dans les SG que ce soit suite à un stress cytoplasmique ou du réticulum endoplasmique. Nous avons montré également que la localisation de la protéine CIRP dans les SG se déroule indépendamment de la présence de la protéine TIA-1 qui a été décrite comme responsable de l’assemblage des SG. De plus la surexpression de la protéine CIRP conduit à la formation de SG. Nous suggérons donc qu’il existe plusieurs voies qui mènent à l’assemblage de ces structures. En outre, nous avons analysé la localisation de mutants par délétion de la protéine CIRP et avons montré que le domaine RRM et le domaine RGG peuvent indépendamment localiser la protéine dans les SG. Par contre, la méthylation des résidus arginine du domaine RGG est une modification nécessaire à la localisation de CIRP dans les SG. Ensuite, nous avons étudié la fonction de la protéine CIRP dans le métabolisme des ARN messagers. Nous avons montré par une méthode d’adressage, que CIRP est un répresseur de la traduction des ARNm et que le domaine carboxy-terminal est nécessaire et suffisant à cette fonction. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished