Excellence in Research: Elucidation of Conserved Eukaryotic Nucleolar and Ribosomal Stress Mechanisms

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This HBCU Excellence in Research award will be used to investigate the biomolecular pathways that link cell growth to the cell cycle and subsequent stress responses that occur when cell growth is arrested in both unicellular and multicellular eukaryotes. Scientific data suggests that unicellular and multicellular eukaryotes utilize different mechanisms to link those processes. Our long-term goal is to elucidate unique and conserved pathways between these species. The yeast data will offer a glimpse into the ancestral stress response mechanisms, and by comparing those data with Drosophila data we will be able to identify conserved as well as modern alterations and evolutionary changes that have occurred since the rise of multicellularity. Our primary scientific question is, “Do unicellular and multicellular eukaryotes harbor similar pathways for coupling cell growth to the cell cycle”, and if so, “What are these pathways?” This basic question has implications in understanding all eukaryote respond to stalled growth arrest and the underlying mechanisms couple that arrest to the cell cycle.Ribosome biogenesis is an essential universal cellular process. Though our knowledge of ribosome biogenesis is quite extensive, there are several unanswered questions regarding stalled or aberrant ribosome biogenesis and how it affects the cell cycle. There is currently a significant contrast between nucleolar and ribosomal stress (NARS) phenotypes in unicellular eukaryotes and metazoans. However, the most frequently observed NARS phenotype is cell cycle arrest. This observation highlights the intimate link between cell growth and the cell cycle and suggest the existence of a common mechanism for this arrest. Our aim is to elucidate the mechanism that cells employ to halt the cell cycle upon repression of ribosome biogenesis. Our experimental design is to replace endogenous yeast ribosomal protein promoters with an inducible GAL1 promoter. With these yeast strains we will systematically express and the repress each of six ribosomal proteins that when repressed lead to a G1 cell-cycle arrest. Using RNA-Seq, we will identify the common differentially expressed genes (DEGS) for each repressed ribosomal protein. In Drosophila, we will use the Tet-on system to systematically express siRNAs that target the same set of ribosomal proteins as described for yeast. As in yeast, we will use RNA-Seq before and after repression of the Drosophila ribosomal proteins and identify the DEGs . We will compare the yeast and Drosophila data and determine the common DEGs within both organisms. This will allow us to identify conserved mechanisms for NARS phenotypes encompassing both unicellular and multicellular eukaryotes. The proposed research will identify novel NARS pathways and significantly advance our understanding of NARS responses.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.