The planned research aims to quantitatively understand the function of key enzymes and their associated complexes in RNA metabolism. We focus on the DEAD-box RNA helicases Ded1p from S. cerevisiae and human DDX3X and their role in eukaryotic translation initiation. This research is critical for understanding functin and regulation of DEAD-box RNA helicases and for illuminating eukaryotic translation initiation. The work is important for delineating the molecular basis of several tumor types and for understanding host- pathogen interactions of multiple infectious diseases. We also investigate the TRAMP-exosome machinery from S. cerevisiae and its role in nuclear RNA metabolism. This line of inquiry is critical for understanding nuclear RNA quality control, regulation of non-coding RNA expression, and nuclear processing of several RNA types. This research has implications for the molecular basis of certain neurodegenerative and autoimmune diseases and for several tumor types. Using a combination of biochemical, biophysical and next generation sequencing approaches, we examine how Ded1p, which interacts promiscuously with a broad range of RNAs, is recruited to and functions at specific sites in the cell. In addition, we determine how post-translational modifications impact the function of Ded1p in vitro and in the cell. For human DDX3X, we establish a mechanistic framework and examine how two viral proteins that interact with the enzyme, HCV core and vaccinia virus K7, affect the function of DDX3X in vitro and in the cell. For the TRAMP-exosome machinery, we determine degradation kinetics at single nucleotide resolution for the entire yeast exosome and quantitatively measure how polyadenylation and decay are coordinated in the TRAMP-exosome machinery. Finally, we examine the function of the TRAMP-exosome machinery on a kinetic level in the cell. We anticipate our work to provide unprecedented quantitative knowledge about the function of enzyme complexes in vitro and in the cell and we expect fundamentally novel insight into eukaryotic translation initiation and nuclear RNA metabolism.
Defects in RNA metabolism have been linked to cancer, viral infections, neurodegenerative disorders and autoimmune diseases. To examine the molecular basis for these diseases and to guide the development of potential therapeutic agents, we propose to delineate the molecular function of RNA helicases and of the TRAMP-exosome machinery, both of which are critical for RNA metabolism and cellular function.
