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JOHNS HOPKINS UNIVERSITY, THE

The long-term objective of this proposal is to understand the role of the RNA polymerase II C-terminal repeat domain (CTD) in transcription regulation during cell growth and quiescence. Understanding this regulatory process will be important in understanding how mis-regulation of CTD function contributes to inappropriate growth leading to cancer. Transcription termination mechanisms controlled by the CTD and its binding proteins may also be involved in the pathogenesis of neurodegenerative diseases. The current proposal is directed at understanding the role of the CTD-binding protein Nrd1 and associated proteins in facilitating transcription termination. This aim will be achieved through an in vitro biochemical approach. We will establish an in vitro termination assay and assess the function of different components and their genetically-modified derivatives. How the termination process is regulated during growth in the yeast Saccharomyces cerevisiae and what transcripts are targets of this regulation will also be investigated. For this part of the project we will employ a combination of yeast genetics and genomic approaches. In particular we will follow the distribution of Nrd1 and RNA polymerase II on the genome during different stages of growth and in different mutant backgrounds. The results of these experiments will allow us to understand how cells prepare for and execute gene regulation in quiescence. Finally, we will identify the targets of the human Nrd1-related protein Rbm16 and test the hypothesis that this protein regulates transcription termination in human cells. This aim will be achieved by mapping the Rbm16 binding sites on RNA. We will also use a proteomic approached to identify proteins that interact with Rbm16. This approach will test the hypothesis that Rbm16 interacts with proteins that are implicated in neurodegenerative disease. These experiments address one of the most fundamental aspects of genetic control. Turning genes on or off as cells make the transition between growth and quiescence is critical to maintaining cell viability in tissues. Loss of growth control can lead to cancer or to cell death depending on the type of cell. This proposal addresses the poorly understood gene regulatory steps that underlie diseases like cancer and neurodegeneration. A firmer understanding of these regulatory processes will aid in development of strategies to cure or prevent a number of human diseases.

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