The regulation of ribosome biogenesis (RB) plays a central role in maintaining cellular homeostasis and supporting cell growth. The rate-limiting step in this process is transcription of the ribosomal RNA genes by RNA polymerase I (Pol I). Dysregulation of RB can contribute to pathologies such as cancer, cardiac hypertrophy, and ribosomopathies. Further, many chemotherapeutic drugs inhibit either rDNA transcription or rRNA processing, but have many off-target effects that limit their usefulness. Many pathways play a role in the regulation of rDNA transcription. Two mammalian factors that are involved in this regulation are Polymerase Associated Factor 53 (PAF53) and PAF49. The purpose of this study is to determine the role(s) of PAF49 and 53 in rDNA transcription and characterize the downstream physiological effects of directly inhibiting this process in both normal and cancer cells. This will be an important comparison since normal cells arrest when rDNA is inhibited while cancer cells die. To rapidly degrade PAF49/53, a novel system that utilizes CRISPR/Cas 9 and an auxin inducible degron will be used. This will allow us to carry out in vitro biochemical and ?genetic? studies of PAF49/53 in mammalian cells. Our published data demonstrates that PAF53 is required for rDNA transcription and cell proliferation. In addition, the three domains of PAF53 are each necessary but not sufficient to support wild-type levels of cell growth. Our lab has also defined a second DNA-binding domain in PAF53 that had not been discovered. This study will expand upon the domain analysis of PAF53 and further characterize its DNA-binding activity. The work proposed is significant as it will aid in further understanding the process of rDNA transcription by Pol I and the physiological consequences of inhibiting this process, i.e. nucleolar stress and cell death. It will also contribute to the discovery of novel drug targets that could be utilized in effective cancer treatments. To complete the proposed research, I will be exposed to new techniques such as crosslinking mass spectrometry, live-cell imaging, and chromatin immunoprecipitation. To improve my scientific communication skills, I will attend and present at the OddPols, AACR and other national and local conferences, as well as attend workshops on scientific writing. I will also prepare multiple manuscripts for publication. Further, I will take multiple opportunities to gain teaching experience. I also will attend seminars and professional development workshops to help extend my scientific purview beyond my field. These opportunities will allow me to network and engage with fellow scientists in order to build connections for future collaborative projects. Overall, the training I will receive during this fellowship will help prepare me to be a competitive postdoctoral candidate and a successful independent research scientist.
Ribosome biogenesis is dysregulated in hyperproliferating cells and represents a novel target for cancer chemotherapy. This project seeks to delineate the biochemical and cellular functions of two essential and unique components of the ribosome biogenesis apparatus in hopes of defining different modalities that regulate ribosome biogenesis and identifying potential therapeutic targets. The proposed study will utilize a model system using CRISPR/Cas 9 and an auxin inducible degron that will allow us to target these two components for rapid degradation so that we can carry out biochemical and ?genetic? studies of these components in mammalian cells.
