Regulatory functions of intrinsically disordered electronegative clusters (ENC) in RNA-binding proteins

生物分子之间的相互作用是生命所必需的。虽然大多数蛋白质具有稳定的三维结构，但有相当数量的蛋白质被证明缺乏稳定的三维结构，被称为内在无序蛋白质。已有研究表明，与RNA相互作用的蛋白质含有本质上无序的区域，这些区域对于蛋白质-RNA结合是必不可少的。该项目的研究人员已经证明，RNA结合蛋白中最丰富的无序区域编码酸性残基或磷酸化位点簇，使这些区域总体上带有负电荷。就像帮助动物平衡和赶走昆虫的尾巴一样，无序的蛋白质区域有助于蛋白质平衡表面电荷以保持稳定性，并迅速赶走非特定配体。这个项目将探索这些负电团是如何调节蛋白质功能的。研究这些蛋白质将提供对关键生物学过程的洞察，如无序蛋白质的瞬时折叠，核糖体生物发生中的位置选择，以及选择性剪接。此外，了解这一现象将有助于开发新的蛋白质设计技术，以获得更强大和特定的RNA结合蛋白质，这最终将有助于美国的生物经济。该项目将为高中生和本科生提供生化研究和生物信息学编码方面的培训机会，包括代表人数不足的群体的成员。本项目将研究三种模型蛋白：组蛋白-mRNA干环结合蛋白、核糖体生物发生蛋白15和富丝氨酸-精氨酸剪接因子1。之所以选择这三种蛋白，是因为它们分别在组蛋白前mRNA加工、核糖体生物发生和选择性剪接等关键生物学过程中发挥重要作用。由于这些蛋白质编码不同类型的电负性簇，并具有独特的三维折叠状态，因此本项目将系统地阐明电负性簇在调节蛋白质折叠和稳定性以及RNA结合特异性和动力学方面的功能。将使用核磁共振、结晶学、停流荧光和下一代测序等尖端生物物理和生化技术。这项工作的完成不仅将产生普遍适用于所有RNA结合蛋白质的知识，还将阐明这些蛋白质发挥关键作用的具体和重要过程。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Interaction between biomolecules is essential for life. While most proteins have stable three-dimensional structures, a significant number of proteins have been shown to lack a stable three-dimensional structure and are known as intrinsically disordered proteins. It has been shown that proteins that interact with RNA contain regions that are intrinsically disordered, which are essential for the protein-RNA binding. The investigators of this project have shown that most abundant disordered regions in RNA-binding proteins encode clusters of acidic residues or phosphorylation sites that give these regions an overall negative charge. Like tails that help animals to balance and to swish away insects, the disordered protein regions help proteins to balance surface charges for stability and swish away nonspecific ligands. This project will explore how these electronegative clusters modulate protein function. Studying these proteins will provide insights into key biological processes, such as transient folding of disordered proteins, site selection in ribosomal biogenesis, and alternative splicing. In addition, understanding this phenomenon will inform development of novel protein design techniques for more robust and specific RNA-binding proteins, which will ultimately contribute to US bioeconomy. This project will provide training opportunities to high school and undergraduate students, including members of underrepresented groups, in biochemical research and bioinformatics-based coding. This project will study three model proteins: histone-mRNA Stem-Loop Binding Protein, ribosome biogenesis protein 15, and Serine-Arginine Rich Splicing Factor 1. These proteins were selected because they play essential roles in critical biological processes, such as histone pre-mRNA processing, ribosomal biogenesis, and alternative splicing, respectively. Because these proteins encode different types of electronegative clusters and have unique degrees of three-dimensional folding status, this project will systematically elucidate the function of electronegative clusters in mediating both protein folding and stability as well as RNA binding specificity and kinetics. Cutting-edge biophysical and biochemical techniques, such nuclear magnetic resonance, crystallography, stopped-flow fluorescence, and next generation sequencing will be used. Completion of this work will not only produce knowledge generally applicable to all RNA-binding proteins, but also elucidate specific and important processes where these proteins play essential roles.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.