Characterizing protein-protein and RNA-protein interactions in fixed cells and tissue
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Blanchard, Emmeline LeGendre
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Abstract
The molecular machinery of the cell is governed by interactions between proteins, DNA, and RNA. For example, innate immune system activation is characterized by the formation and disassociation of protein complexes in a signaling cascade. Alternatively, protein interactions with mRNA control the transcription, transport, and stability of mRNA. Resultingly, abnormal interactions disrupt function, contributing to tumorigenesis. Similarly, protein-protein and protein-RNA interactions in the ribonucleocapsid complex of respiratory syncytial virus govern transcription and replication of the virus.
To gain a better understanding of these interactions, a thorough investigation is required in cells and tissue samples. Conventionally used tools to study interactions are often limited in their specificity and sensitivity. The overall goal of the work presented here was to demonstrate and develop a toolbox to quantify and localize protein-protein and RNA-protein interactions in fixed cells and tissues, and to use these tools to gain insight into the role of molecular interactions in several applications. First, we demonstrated the utility of proximity ligation assays to measure innate immune system activation in both cells and tissue. Next, we developed a methodology of measuring mRNA-protein interactions in archival, patient-derived tissue, specifically examining abnormal mRNA-protein interactions in cancer. Finally, we used proximity ligation assays and our developed methodology to investigate the role of the respiratory syncytial virus ribonucleocapsid complex in the transcription/replication switch in infection. We believe these methods are easy to use and highly translatable to new applications, and therefore of great interest to research where molecular interactions are of importance.
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2019-10-28
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Dissertation