RNA Binding Motif (RBM) domain proteins RBM5 and RBM10 have been shown to influence apoptosis, cell cycle arrest and splicing in transformed cells. In this study, RBM5 and RBM10 were examined in non-transformed cells in order to gain a wider range of knowledge regarding their function. Expression of Rbm5 and Rbm10, as well as select splice variants, was examined at the mRNA and protein level throughout H9c2 skeletal and cardiac myoblast differentiation. Results suggest that Rbm5 and Rbm10 may (a) be involved in regulating cell cycle arrest and apoptosis during skeletal myoblast differentiation and (b) undergo post-transcriptional or translational regulation throughout myoblast differentiation. All in all, the expression profiles obtained in the course of this study will help to suggest a role for Rbm5 and Rbm10 in differentiation, as well as possible differentiation-specific target genes with which they may interact.

Ribonuclease III is a highly-conserved bacterial enzyme that cleaves double-stranded (ds) RNA structures, and participates in diverse RNA maturation and decay pathways. Essential insight on the RNase III mechanism of dsRNA cleavage has been provided by crystallographic studies of the enzyme from the hyperthermophilic bacterium, Aquifex aeolicus. However, those crystals involved complexes containing either cleaved RNA, or a mutant RNase III that is catalytically inactive. In addition, neither the biochemical properties of A. aeolicus (Aa)-RNase III, nor the reactivity epitopes of its cognate substrates are known. The goal of this project is to use Aa-RNase III, for which there is atomic-level structural information, to determine how RNase III recognizes its substrates and selects the target site. I first purified recombinant Aa-RNase III and defined the conditions that support its optimal in vitro catalytic activity. The catalytic activity of purified recombinant Aa-RNase III exhibits a temperature optimum of 70-85°C, a pH optimum of 8.0, and with either Mg2+ or Mn2+ supports efficient catalysis. Cognate substrates for Aa-RNase III were identified and their reactivity epitopes were characterized, including the specific bp sequence elements that determine processing reactivity and selectivity. Small RNA hairpins, based on the double-stranded structures associated with the Aquifex 16S and 23S rRNA precursors, are cleaved in vitro at sites that are consistent with production of the immediate precursors to the mature rRNAs. Third, the role of the dsRBD in scissile bond selection was examined by a mutational analysis of the conserved interactions of RNA binding motif 1 (RBM1) with the substrate proximal box (pb). The individual contributions towards substrate recognition were determined for conserved amino acid side chains in the RBM1. It also was shown that the dsRBD plays key dual roles in both binding energy and selectivity, through RBM1 responsiveness to proximal box bp sequence. The dsRBD is specifically responsive to an antideterminant (AD) bp in pb position 2. The relative structural rigidity of both dsRNA and dsRBD rationalizes the strong effect of an inhibitory bp at pb position 2: disruption of one RBM1 side chain interaction can effectively disrupt the other RBM1 side chain interactions. Finally, a cis-acting model was developed for subunit involvement in substrate recognition by RNase III. Structurally asymmetric mutant heterodimers of Escherichia coli (Ec)-RNase III were constructed, and asymmetric substrates were employed to reveal how RNase III can bind and deliver hairpin substrates to the active site cleft in a pathway that requires specific binding configurations of both enzyme and substrate. / Chemistry

Schizophrenia is a severe psychiatric disorder with unsatisfactory treatment options and poorly under- stood etiology. Genetic models are a suitable tool for studying this disorder with its high heritability. However, currently available animal models do not cover the broad range of schizophrenia symptoms and are not disorder-specific. Ribonucleic acid binding motif protein 12 gene (RBM12), a novel, high- risk gene for schizophrenia, was recently identified. This thesis aimed to assess the social behavior of schizophrenia-like phenotype in RBM12 zebrafish mutants. The social behavior of mutated adult zebrafish was assessed during free-swimming. Trajectories of each zebrafish were obtained from recordings by the usage of idtracker.ai. Parameters selected to quantify the social behavior of the zebrafish were chosen based on common symptoms of humans with schizophrenia. Inter-fish distance was examined as an indicator of preferred personal space since humans diagnosed with schizophre- nia have an increased need for a greater personal space compared to mentally healthy individuals. Wall-hugging, increased speed and bottom-dwelling were studied as indicators of anxiety, a common comorbid symptom of schizophrenia. The RBM12 mutants exhibited a greater inter-fish distance than their wild-type siblings during three-dimensional recordings. They however, did not demonstrate an increased inter-fish distance during two-dimensional recordings. The mutated zebrafish displayed a higher average speed and greater wall-hugging, indicating anxiety. It can be concluded that RBM12 mutation produces partial symptomatology consistent with humans diagnosed with schizophrenia, providing a promising animal model. The current work provided novel insight into the neural substrates of schizophrenia and for potential drug screening for this disorder. Further research is needed to fully characterise schizophrenia-like symptoms in this RBM12 animal model.