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No description available.

No description available.

No description available.

No description available.

No description available.

CELF4 is a brain-specific member of the CELF RNA binding protein (RBP) family that binds a significant portion of the transcriptome with striking selectivity for the 3’UTR of neuronal and synapse-specific functional targets in the hippocampus. Celf4 knockout and haploinsufficient mice have a complex neurobehavioral phenotype similar to human patient groups identified with CELF4 mutations, specifically CELF4-inclusive deletions and translocations. We hypothesize that CELF4 operates in multiple aspects of post-transcriptional gene regulation; interacting with RNA molecules from synthesis to decay. Tissue-level ribosome profiling experiments demonstrate that loss of CELF4 results in global ribosome occupancy changes across CELF4 mRNA targets and refined our ability to interrogate the synaptic function of CELF4. Turning intra-cellularly, a snRNA-seq approach implicated the CA3 region of the hippocampus in CELF4-mediated mRNA regulation and identified synaptic targets regulated by CELF4. By leveraging both ribosome profiling footprinting and snRNA-seq differential gene expression data, we identified synaptic and epilepsy disease genes that contribute to, and drive, neurobehavioral phenotypes. In part two of this work we focus on DEE disease gene DNM1, a known target of CELF4 at the synapse. Using in vitro and in vivo approaches, we validate the regulatory relationship between mouse Dnm1 RNA and CELF4 RBP function. Lastly, we introduce a novel preclinical model of DNM1 DEE that recapitulates the seizure and behavioral phenotypes of patients suffering from dominant negative DNM1 mutations. In characterization of this model, we lay the groundwork for future investigations of cellular etiology of DNM1 pathogenic variants and therapeutic development for patient groups suffering from DEE.

Maintaining genomic integrity is important for any organism. DNA mismatch repair (MMR) serves to correct errors that occur during DNA replication and recombination, such as unpaired bases or mismatched bases. Mutl is a key player and serves to coordinate protein-protein interactions. Recently it has been shown that human Mutl functions as an endonuclease and that this activity is imperative for functioning MMR. In this work, the X-ray crystal structure of the C-terminal endonuclease domain of Bacillus subtilis Mutl (BsMutL-CTD) is presented. Diffraction quality crystals of BsMutL-CTD were grown using vapor diffusion. The crystal structure of BsMutL-CTD was solved using multiwavelength anomalous diffraction. The structure reveals a putative metal binding site which clusters closely in space with endonuclease motif. Using the structure and sequence homology, several mutations were made and an investigation into the endonuclease activity of BsMutL was performed. BsMutL was confirmed to be a manganese-dependent endonuclease and key residues which contribute to endonuclease function were identified. / Thesis / Master of Science (MSc)

Adherens junctions are a main cell-cell adhesion structure found in epithelial cells. The stability of adherens junctions is attributed to various protein-signaling cascades and importantly the interaction between the transmembrane protein E-cadherin and cytoplasmic p120 catenin. This interaction is critical for cell adhesion and prevention of uncontrolled growth in normal cells. The interaction interface between these two binding partners was previously determined to comprise p120's Armadillo repeat domain (p120Arm) and Ecadherin's cytoplasmic juxtamembrane domain (Ecadc). Based on this information, peptide aptamers were derived from p120Arm and their interaction with Ecadc was tested in vitro. We reasoned that those could be expressed in vivo to stabilize adherens junctions at the cell-cell junction. In this study, we established protein-protein interaction assays to demonstrate p120Arm's ability to bind Ecadc and then used these assays to determine if p120Arm-derived peptides may competitively bind Ecadc. We demonstrated the interaction between p120Arm and Ecadc using assays that were not previously used such as: co-precipitation, analytical gel filtration and the bacterial-2-hybrid assay. However, the p120Arm-derived peptides did not bind to Ecadc or compete its interaction with p120Arm. This may be due to the nature of the assays that may not reflect competitive binding or the aptamers may not adopt the native conformation preventing binding to Ecadc. / Thesis / Master of Science (MSc)

The ability of neurons to send and receive signals underlies the most essential functions of the brain. Thus, the formation and function of new synapses must be tightly regulated. Local protein synthesis is essential for presynaptic terminal formation and persists at mature synapses. We have recently discovered a role for Pumilio 2 as a negative regulator of axonal localization and translation of its target mRNAs. Pumilio RNA-binding proteins regulate a large number of synaptic mRNAs encoding proteins essential for neurotransmission and neuron projection development and are developmentally downregulated in the brain, corresponding with the increased translation of their target mRNAs in axons. Here, I tested the hypothesis that Pumilio proteins constrain the formation and/or maturation of synapses at early stages of neuronal maturation by regulating synaptic mRNAs. I found that simultaneous downregulation of Pumilio 1 and 2 together induces an increase in synapses in primary hippocampal neurons, while downregulation of Pumilio 1 or Pumilio 2 individually results in a reduction in synapse density. The increase in synapses seen with dual Pumilio knockdown corresponds with an increase in both excitatory and inhibitory presynaptic markers as well as an increase in Snap25 translation. Notably, this increase in synapses persists even when Pumilios are knocked down at later stages of maturation after developmental downregulation has already occurred. This suggests that remaining low levels of Pumilio proteins continue to play a significant role in plasticity and regulation of the synapse at later stages of neuronal maturation, potentially throughout the lifespan of an organism.