Exploring Tissue-Specific Mechanisms and Requirements for Nucleolin Organization in Vivo - PROJECT SUMMARY Precise organization of cellular space is a fundamental theme of eukaryotic cell biology and is essential for control over complex biological reactions. Subcellular organization of RNA binding proteins enables targeted regulation of RNA metabolism, and their disorganization is a hallmark of human disease. For example, the RNA binding protein, Nucleolin (NCL), displays precise organization within the nucleolus. This organization is considered critical for ribosome biogenesis and, consequently, is disrupted in several forms of disease. Because NCL is an essential gene in vertebrate animal models and no clear homolog exists in Drosophila, most studies of NCL organization use in vitro models. However, the nucleolus is exquisitely sensitive to environment and in vitro conditions can influence nucleolar dynamics. As a result, the biological mechanisms that regulate NCL organization are unclear. We surmount these challenges with our discovery of the C. elegans homolog of NCL, named NUCL-1. Deleting endogenous NUCL-1 results in viable worms, providing the opportunity to fill a large gap in knowledge and explore mechanisms of NCL organization in a living animal, to better understand its role in disease. C. elegans are short-lived, optically clear animals with facile genetics, making them the ideal model for our proposed in vivo studies. We recently used CRISPR/Cas9 genetic engineering to develop an in vivo, split- fluor system to visualize NUCL-1 in germ cell nucleoli. Combining this system with super-resolution (AiryScan) imaging, we were the first to map the intricate sub-nucleolar organization of germ cells in living C. elegans. Precise sub-nucleolar organization is thought to be essential for ribosome biogenesis in all eukaryotic cells, but our in vivo work challenges this hypothesis. Full deletion of the nucl-1 gene causes impaired fertility, delayed development, extended lifespan, and smaller body size, all phenotypes linked to impaired ribosome biogenesis. In contrast, deleting the arginine-glycine (RG) repeat domain from endogenous NUCL-1 disrupts sub-nucleolar organization, but worms are healthy and fertile with motor hyperactivity. These results decouple precise sub- nucleolar organization from phenotypes associated with ribosome biogenesis and lead to the hypothesis that there are tissue-specific requirements for sub-nucleolar organization. Over the next five years, we will expand our in vivo, split-fluor system into a modular genetic toolkit with which to extend our study of NUCL-1 organization to all major somatic tissues. Using this toolkit we will address the following knowledge gaps: (1) What is the extent of diversity in sub-nucleolar organization across somatic tissues and how does the NUCL-1 RG repeat domain contribute to that organization, and (2) Which somatic tissues require precise sub-nucleolar organization for their normal function? The proposed research will explore tissue-specific patterns, mechanisms, and functions of sub-nucleolar organization and will lay the groundwork for understanding the role of NCL organization in human disease.
