Nab2, Molecular Recognition of Polyadenosine RNA by a Zinc Finger Protein

这个项目，这是在生物学和物理学的界面，检查一个非常常见的大分子相互作用领域，锌指motif。锌指蛋白是真核生物中含量最多的蛋白之一，在许多生物过程中起着至关重要的作用。研究人员使用了一种全面的方法来了解一个进化上保守的锌指蛋白家族，包括含有串联（CCCH）锌指的酿酒酵母Nab2蛋白，是如何与RNA相互作用的。Nab2是一种重要的酵母蛋白，在mRNA加工和mRNA从细胞核向细胞质的输出中起着关键作用。先前的研究表明，Nab2锌指结构域是mRNA结合所必需的，初步数据表明其优先与多腺苷RNA结合。将进行三个领域的研究：1)确定Nab2和ZC3H14中存在的锌指基序是否确实赋予序列特异性结合聚(A) RNA；2)定义多个锌指如何促进结合特异性和/或高亲和力核酸结合，从而深入了解串联锌指如何赋予序列特异性结合聚(A) RNA；3)利用Nab2锌指突变体（C437S）的RNA结合减少，了解体内RNA结合的需求，并确定调节Nab2与mRNA转录物相互作用的因素。这项研究的智力价值是双重的：1)将获得关于锌指蛋白新家族如何识别RNA的见解；2)发展通常不用于研究蛋白质核酸相互作用的生物物理方法。研究人员的方法采用了荧光相关光谱（FCS）、生化方法和酵母的遗传研究。所描述的研究是Corbett（埃默里医学院生物化学系）和Berland（埃默里学院物理系）实验室的合作成果。因此，这些研究处于生物学和物理学的交叉点。这项研究产生的更广泛的影响是对培养本科生，研究生研究人员和博士后研究员的重大贡献，包括通过物理和生物学受训人员的重点互动进行跨学科培训。重要的是，这些研究也为课堂上跨学科学习的发展奠定了基础，并为与埃默里大学以外的社区交流提供了更多的机会。PI的两个实验室都与包括亚特兰大地区的高中学生在内的各个层次的学生有长期的互动历史，通过在实验室接待学生和老师。拟议的研究将加强与对物理学/生物物理学感兴趣的学生的互动，并有力地说明跨学科研究的力量。这项研究的目标是了解细胞遗传物质中的信息实际上是如何被读取并用作蓝图，以创建制造和维持细胞所需的构建块。研究人员将研究信使分子RNA，它将信息从细胞核转移到细胞质中，在细胞质中存在着实际翻译遗传信息的机制。这个过程被称为信使RNA输出，是基因表达或读取遗传密码的关键步骤。这项工作结合了生物化学和物理学，从一个新的方向来解决这个重要的问题，也开发了以前没有用于研究这个问题的方法。大部分工作包括本科生，他们在生物实验室和物理实验室之间共同工作。生物学和物理学之间的这种接口也允许研究人员开发新的训练方法，包括跨学科课程和实验室。

This project, which is at the interface of biology and physics, examines a very common macromolecular interaction domain, the zinc finger motif. Zinc finger proteins, which are among the most abundant proteins in eukaryotes, play critical functions in many biological processes. The researchers use a comprehensive approach to understand how an evolutionarily conserved family of zinc finger proteins including, the Saccharomyces cerevisiae Nab2 protein, which contains tandem (CCCH) zinc fingers, interacts with RNA. Nab2 is an essential yeast protein that plays critical roles in both mRNA processing and mRNA export from the nucleus to the cytoplasm. Previous studies have demonstrated that the Nab2 zinc finger domain is required for mRNA binding and the preliminary data suggests preferential binding to polyadenosine RNA. Three areas of research will be pursued: 1) determining whether the zinc finger motifs present in Nab2 and ZC3H14 do indeed confer sequence specific binding to poly(A) RNA; 2) defining how multiple zinc fingers contribute to binding specificity and/or high affinity nucleic acid binding thus providing insight into how tandem zinc fingers confer sequence-specific binding to poly(A) RNA; and 3) exploiting a zinc finger mutant of Nab2 (C437S) with documented decreased RNA binding to understand the requirement for RNA binding in vivo and to identify factors that regulate the interaction of Nab2 with mRNA transcripts. The Intellectual Merit of the research is two-fold: 1) the insights that will be gained into how a novel family of zinc finger proteins recognizes RNA; and 2) the development of biophysical methods not typically employed to study protein nucleic acid interactions. The researchers' approach employs Fluorescence Correlation Spectroscopy (FCS), biochemical approaches, and genetic studies in yeast. The studies described are the collaborative effort of the Corbett (Biochemistry Department, Emory School of Medicine) and the Berland (Physics Department, Emory College) laboratories. Thus, these studies lie at the interface of biology and physics. The Broader Impacts resulting from this research are significant contributions to training undergraduate students, graduate researchers, and postdoctoral fellows, including cross-disciplinary training through the focused interactions of trainees in physics and biology. Importantly, these studies also set the stage for the development of interdisciplinary learning in the classroom and afford enhanced opportunities for interface with the community beyond Emory. Both PI's laboratories have a long-standing history of interaction with students at all levels including those in high schools in the Atlanta area through hosting both students and teachers in the lab. The proposed studies would enhance interactions with students interested in Physics/Biophysics in and provide a strong illustration of the strength of interdisciplinary research.The goal of this research is to understand how information within the genetic material of cells is actually read and used as a blueprint to create the building blocks needed to make and maintain cells. The researchers will study the messenger molecule, RNA, that moves the information from the cell nucleus out to the cell cytoplasm where the machinery is present to actually translate the genetic information. This process, called messenger RNA export is a critical step in gene expression or reading the genetic code. The work combines biochemistry and physics to approach this important question from a new direction and also to develop methods not previously used to study this question. Much of the work includes undergraduate students who work jointly between a Biological laboratory and a Physics laboratory. This interface between Biology and Physics also allows the researchers to develop new training methods including interdisciplinary courses and laboratories.