lluminating the biochemistry of zinc and RNA in live cells

项目摘要 锌是仅次于铁的哺乳动物体内第二丰富的过渡金属。有两千多个 由人类基因组编码的含有锌结合基序的蛋白质，其中锌结合被预测为 对功能是必不可少的。在细胞水平上，锌对DNA合成、细胞增殖、分化、 在生物体水平上，锌是生长、发育和免疫功能所必需的。 鉴于锌在细胞生物学和人类健康中的重要性，令人震惊的是，我们仍然不知道 锌离子水平和动力学如何影响基本细胞功能和引发疾病的机制。 尽管生物学上对锌的传统观点认为它是结构性的和稳定地结合在蛋白质上， 包括锌蛋白质组在内，越来越多的证据表明细胞内的锌离子是动态的。此外，我们的实验室已经 表明锌离子的动力学深刻地影响着基本的细胞过程，如转录、分泌 途径功能，以及细胞周期，坚定地确立了锌离子是一种信号离子。然而，这些蛋白质和 感知锌离子动力学以影响细胞变化的途径仍然是一个谜。我的研究项目是 准备通过探索锌离子动力学滴定占有率从而解决这个问题的假设来解决这个问题 锌离子蛋白质组的活性。因此，在生理信号、环境和环境中锌离子的变化 干扰，或作为疾病的结果-可能微调数千锌依赖的活动 蛋白质，使锌离子成为细胞功能的主要调节因子。我们通过以下方式解决这一假设 解决4个主要问题：(1)锌蛋白质组中哪些蛋白质感知锌离子的动态变化 现状？(2)锌通过调节转录因子的滴定功能和DNA结合来调节转录吗？(3) 有哪些途径和蛋白质介导了锌离子对哺乳动物细胞周期的调控？以及(4)如何 缺乏锌是否会影响其他必需金属(铁、铜和锰)的调节？要解决这些问题 问题，我们将使用基因组学、化学蛋白质组学、活细胞成像和生物化学的组合 接近了。最近，我的实验室利用了我们在工具开发、生物物理和光物理方面的专业知识 荧光探针的表征和活细胞测量的分析方法，以开发一种新的 用荧光团标记mRNA和ncRNA的平台，以在活细胞中跟踪它们。该平台填补了一个 重要的技术需求，因为有诱人的建议表明RNA本地化与 动态和功能，但在现有的工具库中存在重大限制。因此，有一个 迫切需要强大的、互补的和最小干扰的工具来可视化单个RNA分子 在活细胞中绘制复杂和不断演变的RNA生物学图景。因此，最后一个组件是 我的研究计划是(5)满足对标记和跟踪活细胞中RNA的改进工具的技术需求。 具体地说，我们将开发一套基于核糖开关的RNA标签，用于绑定模块化化学探针。我们会 还开发了一系列可靠的分析方法，用于在活细胞中对RNA标记工具的性能进行基准测试。

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.