This project studies basic principles that govern organization of the cell nucleus. The nucleus has a special importance for biotechnology because it stores genetic information that controls traits of cells and organisms. Continuous success of biotechnology greatly depends on better understanding of the processes inside of the nucleus. This project will specifically focus on studying the sorting mechanism that enables certain proteins to form distinct and isolated structures within the nucleus. In a separate set of experiments, the information learned in this project will be applied to improve the current biotechnology methods. The desired outcome is to make genetic manipulations more efficient and safer. As a broader impact, this project will offer an educational platform at Kennesaw State University (KSU) to train undergraduate and graduate students with contemporary methods of molecular biology and genetic engineering. KSU is a dynamically developing, predominantly undergraduate institution that strives to enhance its research environment and expand graduate programs.
In the absence of internal membranes in the nucleus, nuclear proteins are organized into compact, distinct areas with sharp boundaries that are called nuclear domains (NDs). The regulation and functional significance of protein sorting into NDs is not clear. This project will use a newly discovered Drosophila ND called a B-body and its resident RNA-binding protein called Bruno, as a model to study ND assembly and function. It is hypothesized that a combination of Bruno’s protein sequence and non-coding RNA is sufficient to create B-bodies. Molecular biology techniques, including mutagenesis and in situ RNA hybridization, will be applied to identify the essential components to reconstitute B-bodies. Next, B-bodies will be tested for their role as safe overflow sites for ectopically expressed nuclear proteins. Heterologous proteins including DNA enzymes and transcription factors will be targeted to B-bodies. Biochemical assays and genetic reporters will quantitatively indicate whether protein trafficking into B-bodies can reduce non-specific effects and toxicity that often plague overexpression experiments.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
