The regulation of mRNA is critical to cellular function. Over the years of this grant we discovered that an important mode of regulation is the ability of messenger RNA to become localized at specific regions in cells where synthesis of specific proteins can be spatially compartmentalized. This has profound implications for cell structure and function since all cells have characteristic shapes and structure, which are essential for them to perform their function. The best example of this is the neuron, where the synapses are very far away from the cell body, sometimes meters away. Messenger RNA may have to travel these far distances and then be activated to make proteins upon stimulation of specific synapses at a precise moment. This is the basis of learning and memory. The mechanism by which the mRNA can remain quiescent for long periods of time and then become active upon a specifically localized stimulus is unknown.
The Specific Aims of this proposal are directed toward developing new technology to address this question. We have made significant experimental, technical and conceptual advances that have allowed us to observe single molecules of mRNA. For instance we have made a mouse where every molecule of the ss-actin mRNA, which makes an essential protein is labeled. This will allow us to observe these molecules in living cells and tissues. We found that the mRNA travels to distant regions of the cell because of a sequence known as the zipcode. We discovered that this sequence binds a protein, the zipcode binding protein (ZBP1) and this binding silences the mRNA until it reaches its final destination. To activate the mRNA to translate, the protein must be modified at its destination by a kinase that phosphorylates it at a specific site. We believe ZBP1 is the key to understanding the regulatory events that occur at specific locations in the cell, for instance at the synapse, where ss-actin is necessary for stabilizing spines important for their presentation to incoming signals. In support of the importance of this protein, if we delete it in mice, the result is lethal, newborn mice do not survive and their brains show defects in organization of the neuronal layers. We have shown that the protein is essential for proper migration of cells, such as fibroblasts and neurons, and this we believe is due to the ability of the cell to direct the synthesis of actin in a polarized location, where it can polymerize and drive the extension of cell structures involved in movement. ZBP1 is also implicated in disease prevention. After birth, the expression of ZBP1 is repressed, but we have been able to make a transgenic mouse that expresses ZBP1 exogenously in the brain and show that these mice have profoundly altered behavior: they become resistant to drug addiction. Furthermore, expression of ZBP1 in the mammary gland makes the mice resistant to breast cancer metastasis. We have also recently discovered that the ZBP1 family of proteins (there are three) is also implicated in preventing neurological diseases and diabetes. Hence not only will study of this protein reveal how mRNA is regulated, but also how disruption of this regulation can lead to a broad variety of diseases.
The mechanism controlling the time and place where messenger RNA is activated to make proteins is unknown, yet this problem is at the heart of all cell function and at the basis of neurological diseases and cancer. We have devised a variety of technologies to answer this question, using transgenic animals, innovative microscopy and unique mRNA reporters to determine where the critical events occur in the cell. We will use this methodology on neurons, where the local activation of protein synthesis at synapses is at the basis of learning and memory.
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