Project Summary Zinc (Zn2+) is the second most abundant transition metal in mammals after iron. There are over two thousand proteins encoded by the human genome that contain zinc binding motifs, where zinc binding is predicted to be essential for function. At the cellular level zinc is important for DNA synthesis, cell proliferation, differentiation, and apoptosis, while at the organism level zinc is required for growth, development and immune function. Given the importance of Zn2+ in cell biology and human health, it is astounding that we still don?t understand the mechanisms of how Zn2+ levels and dynamics impact basic cellular functions and give rise to disease. Although the conventional view of Zn2+ in biology is that it is constitutively and stably bound to the proteins that comprise the zinc proteome, there is growing evidence that Zn2+ in cells is dynamic. Further, our lab has shown that Zn2+ dynamics profoundly influence fundamental cellular processes such as transcription, secretory pathway function, and the cell cycle, firmly establishing that Zn2+ is a signaling ion. However, the proteins and pathways that sense Zn2+ dynamics to effect cellular change remain a mystery. My research program is poised to tackle this question by exploring the hypothesis that Zn2+ dynamics titrate occupancy and hence activity of the Zn2+ proteome. Thus, changes in Zn2+ ? during physiological signaling, environmental perturbation, or as a consequence of disease ? could fine-tune the activity of thousands of zinc-dependent proteins, establishing Zn2+ as a major regulator of cellular function. We are addressing this hypothesis by tackling 4 overarching questions: (1) Which proteins across the zinc proteome sense dynamic changes in Zn2+ status? (2) Does zinc regulate transcription by titrating function and DNA-binding of transcription factors? (3) What are the pathways and proteins that mediate Zn2+ regulation of the mammalian cell cycle? And (4) how does Zn2+ deficiency influence the regulation of other essential metals (Fe, Cu and Mn). To tackle these questions, we will use a combination of genomics, chemical proteomics, live cell imaging, and biochemistry approaches. Recently, my lab exploited our expertise in tool development, biophysical and photophysical characterization of fluorescent probes, and analytical approaches to live cell measurements, to develop a new platform for tagging mRNA and ncRNA with fluorophores to track them in live cells. This platform fills an important technological need, as there are tantalizing suggestions of connections between RNA localization, dynamics and function, but there are major limitations in the existing repertoire of tools. Thus, there is a pressing need for robust, complementary, and minimally perturbing tools to visualize individual RNA molecules in living cells to map the complex and evolving landscape of RNA biology. Therefore, the final component of my research program is to (5) Meet the technological need for improved tools to tag and track RNA in live cells. Specifically, we will develop a suite of riboswitch-based RNA tags that bind modular chemical probes. We will also develop a series of robust assays for benchmarking the performance of RNA tagging tools in live cells.

R35GM139644

2021-01-01

2025-12-31