One third of the world's population is infected with Mycobacterium tuberculosis, the bacterium that causes TB. During an infection, bacteria often survive host immune system attacks, which include oxidative stress conditions for bacteria growing inside macrophages. This makes treatment difficult and time-consuming. We hypothesize bacteria can adapt to environmental conditions by changing their mRNA maturation and degradation profiles. Using a model system, Mycobacteruim smegmatis, we focus on how mRNA expression is affected by oxidative stress. After construction and sequencing of RNA expression libraries, preliminary analysis showed that after three hours of H2O2 exposure most upregulated genes were related to DNA repair, while downregulated genes included transport proteins. After six hours of exposure, upregulated genes were similar to three hours and downregulated genes included tRNAs. 5' end mapping libraries were also constructed to access differential cleavage site abundance under oxidative stress conditions. We also investigated the roles RNase J may have in stress response and mRNA processing in Mycobacteria. RNase J and RNase E are thought to be the major RNases in bacteria. While most bacteria only have one of them, mycobacteria encode both in their genome, with RNase J being non-essential. We constructed a set of 4 strains (WT, RNase J overexpression, RNase J deletion, and complemented RNase J deletion) and tested their drug resistance and stress tolerance. Results suggests that RNase J deletion and overexpression alter drug sensitivity. Stress tolerance assays showed that WT is more tolerant to oxidative stress, followed by RNase J deletion strain and overexpression and complemented RNase J deletion strains, with the last two showing no growth when cultured with H2O2. Analysis of the expression profile of these strains was performed to help understand if gene expression differences are responsible for the phenotypes observed. For the complemented RNase J deletion, one operon had almost all its genes upregulated. This operon encodes a hydrogenase (Hyd3), suggesting that redox balance in the strain is perturbed.

Genome regulation is an extremely complex phenomenon. There are various mechanisms in place to ensure smooth performance of the organism. Post-transcriptional regulation of gene expression is one such mechanism. Many proteins bind to mRNAs and regulate their translation. In this thesis, I have focused on the Cytoplasmic Polyadenylation Element Binding family of proteins (CPEB1-4); a group of sequence specific RNA binding proteins important for cell cycle progression, senescence, neuronal function and plasticity. CPEB protein binds mRNAs containing a short Cytoplasmic Polyadenylation Element (CPE) in 3’ untranslated Region (UTR) and regulates the polyadenylation of these mRNAs and thereby controls translation. In Chapter II, I have presented my work on the regulation of mitochondrial function by CPEB. CPEB knockout mice have brain specific defects in mitochondrial function owing to a reduction in Electron transport chain complex I component protein NDUFV2. CPEB controls the translation of this NDUFV2 mRNA and thus affects mitochondrial function. A consequence of this reduced bioenergetics is reduced growth and branching of neurons, again emphasizing the importance of this pathway. Chapter III focuses on the role of CPEB4 in neuronal survival and protection against apoptosis. CPEB4 shuttles between nucleus and cytoplasm and becomes nuclear in response to stimulation with ionotropic glutamate receptors, focal ischemia in vivo and when cultured neurons are deprived of oxygen and glucose; nuclear CPEB4 affords protection against apoptosis in ischemia model. The underlying cause for nuclear translocation is reduction in Endoplasmic Reticulum calcium levels. These studies give an insight into the function and dynamics of these two RNA binding proteins and provide a better understanding of cellular biology.

Thesis (Ph.D. in Biochemistry) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 135-145). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

The central dogma of biology asserts that DNA is transcribed into RNA and RNA is translated into protein. However, this overtly simplistic assertion fails to portray the highly orchestrated and regulated mechanisms of transcription and translation. During the process of transcription, RNA provides the template for translation and protein synthesis as well as the structural and sequence specificity of many RNA and protein-based machines. While only 1-5% of the genome will escape the nucleus to be translated as mRNAs, complex, parallel, highly-conserved mechanisms have evolved to regulate specific mRNAs. Trans-acting factors bind cis-elements in both the 5" and 3" untranslated regions of mRNA to regulate their stability, localization, and translation. While a few salient examples have been elucidated over the last few decades, mRNA translation can be reversibly regulated by the shortening and lengthening of the 3" polyadenylate tail of mRNA. CPEB, an important factor that nucleates a complex of proteins to regulate the polyadenylate tail of mRNA, exemplifies a major paradigm of translational control during oocyte maturation and early development. CPEB function is also conserved in neurons and somatic foreskin fibroblasts where it plays an important role in protein synthesis dependent synaptic plasticity and senescence respectively. Focusing on the function of CPEB and its role in mRNA polyadenylation during human cellular senescence, the following dissertation documents the important finding that CPEB is required for the normal polyadenylation of p53 mRNA necessary for its normal translation and onset of senescence. Cells that lack CPEB have abnormal levels of mitochondria and ROS production, which are demonstrated to arise from the direct result of hypomorphic p53 levels. Finally, in an attempt to recapitulate the model of CPEB complex polyadenylation in human somatic cells, I unexpectedly find that Gld-2, a poly(A) polymerase required for CPEB-mediated polyadenylation in Xenopus laevis oocytes, is not required for p53 polyadenylation, but instead regulates the stability of a microRNA that in turn regulates CPEB mRNA translation. Furthermore, I demonstrate that CPEB requires Gld-4 for the normal polyadenylation and translation of p53 mRNA.

Local protein synthesis is required for long-term memory formation in the brain. One protein family, Cytoplasmic Polyadenylation Element binding Protein (CPEB) that regulates protein synthesis is found to be important for long-term memory formation possibly through regulating local protein synthesis in neurons. The well-studied member of this family, CPEB1, mediates both translational repression and activation of its target mRNAs by regulating mRNA polyadenylation. Mouse with CPEB1 KO shows defect in memory extinction but not long-term memory formation. Three more CPEB1 homologs (CPEB2-4) are identified in mammalian system. To test if CPEB2-4 may have redundant role in replacing CPEB1 in mediating local protein synthesis, the RNA binding specificity of these homologs are studied by SELEX. The result shows CPEB2-4 bind to RNAs with consensus sequence that is distinct from CPE, the binding site of CPEB1. This distinction RNA binding specificity between CPEB1 and CPEB2-4 suggests CPEB2-4 cannot replace CPEB1 in mediating local protein synthesis. For CPEB2-4 have distinct RNA binding specificity compared to CPEB1, they are referred as CPEB-like proteins. One of CPEB-like protein, CPEB3, binds GluR2 mRNA and represses its translation. The subcellular localization of CPEB family proteins during glutamate over stimulation is also studied. The CPEB family proteins are identified as nucleus/cytoplasm shuttling proteins that depend on CRM1 for nuclear export. CPEB-like proteins share similar nuclear export ciselement that is not present in CPEB1. Over-stimulation of neuron by glutamate induces the nuclear accumulation of CPEB family proteins possibly through disrupted nuclear export. This nuclear accumulation of CPEB family protein is induced by imbalance of calcium metabolism in the neurons. Biochemical and cytological results suggest CPEB4 protein is associated with ER membrane peripherally in RNA independent manner. This research provides general description of biochemical, cytological properties of CPEB family proteins.