Gap junctions, comprised of connexin proteins, are essential for direct intercellular electrical, metabolic, and immunological coupling. Connexin43 (Cx43, gene name GJA1) is the most ubiquitously expressed gap junction protein, and Cx43 gap junctions are altered in pathological states including cardiac disease and cancer. The GJA1 mRNA undergoes alternative translation initiation to yield a truncated Cx43 isoform, GJA1-20k, that can regulate gap junction formation. Using epithelial-mesenchymal transition (EMT) as a cellular model of gap junction remodeling, we have demonstrated altered translation initiation of Gja1 as a mechanism by which cellular Cx43 gap junctions can be dynamically regulated. Suppression of Gja1 alternative translation is necessary for Cx43 gap junction loss, and stable expression of GJA1-20k rescues gap junction formation during EMT. To identify regulatory factors acting on the Gja1 mRNA, an MS2 RNA aptamer tagging system was adapted to isolate Gja1 with associated RNA binding proteins. We find the RNA binding protein IMP1 is sensitive to hypoxic stress and complexes with Gja1 mRNA, where it is necessary for alternative translation to generate GJA1-20k. We have demonstrated alterations in translation initiation of the Gja1 mRNA as a critical mechanism by which cells modulate Cx43 gap junctional coupling in changing conditions and identified a novel regulator of this process in mammalian cells. / Doctor of Philosophy / Communication between cells is necessary for healthy function of organs throughout the body. Gap junctions form conduits through which signals can pass directly between neighboring cells. Many diseases, including cancer and heart disease, involve disturbances in gap junction communication. Connexin proteins are the building blocks of gap junctions, and it was recently demonstrated that smaller fragments of connexins are synthesized by cells by a poorly understood process called alternative translation. Importantly, levels of these connexins fragments can alter gap junction formation. We have used mammalian cells to delineate the mechanism by which this alternative protein translation regulates gap junction formation and generated insight into how such protein synthesis is dynamically regulated. Harnessing this knowledge will inform development of new therapeutics inducing alternative translation to rescue gap junctions, and restore normal communication in pathological conditions.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/89090 |
| Date | 22 April 2019 |
| Creators | James, Carissa Chey |
| Contributors | Graduate School, Smyth, James W., Gourdie, Robert G., McDonald, Sarah, Lamouille, Samy, Mukherjee, Konark |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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