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

这项HBCU卓越研究奖将用于研究将细胞生长与细胞周期联系起来的生物分子途径，以及在单细胞和多细胞真核生物中，当细胞生长受阻时发生的随后的应激反应。科学数据表明，单细胞和多细胞真核生物利用不同的机制来连接这些过程。我们的长期目标是阐明这些物种之间独特和保守的途径。酵母的数据将提供对祖先应激反应机制的一瞥，通过将这些数据与果蝇的数据进行比较，我们将能够识别自多细胞生物兴起以来发生的保守和现代变化和进化变化。我们的主要科学问题是，“单细胞和多细胞真核生物是否有类似的途径将细胞生长与细胞周期结合起来？”如果有，“这些途径是什么？”这个基本问题对理解所有真核生物对生长停滞的反应以及细胞周期停滞的潜在机制具有重要意义。核糖体生物发生是一个重要的普遍细胞过程。尽管我们对核糖体生物发生的了解相当广泛，但关于核糖体生物发生的停滞或异常以及它如何影响细胞周期，仍有几个悬而未决的问题。目前，在单细胞真核生物和后生动物中，核仁和核糖体应激（NARS）表型之间存在显著差异。然而，最常见的NARS表型是细胞周期阻滞。这一观察结果突出了细胞生长和细胞周期之间的密切联系，并表明存在这种抑制的共同机制。我们的目的是阐明细胞在抑制核糖体生物发生时用来停止细胞周期的机制。我们的实验设计是用可诱导的GAL1启动子替代内源性酵母核糖体蛋白启动子。利用这些酵母菌株，我们将系统地表达和抑制六种核糖体蛋白中的每一种，当被抑制时导致G1细胞周期阻滞。使用RNA-Seq，我们将确定每个抑制核糖体蛋白的共同差异表达基因（DEGS）。在果蝇中，我们将使用Tet-on系统来系统地表达针对与酵母相同的核糖体蛋白集的sirna。与酵母一样，我们将在果蝇核糖体蛋白抑制前后使用RNA-Seq来鉴定deg。我们将比较酵母和果蝇的数据，确定这两种生物体内的共同deg。这将使我们能够确定包括单细胞和多细胞真核生物在内的NARS表型的保守机制。拟议的研究将确定新的NARS通路，并显著推进我们对NARS反应的理解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.