CAREER: Structural aspects of glutamine-rich domain liquid-liquid phase separation in transcription and RNA processing

生命体通过创造数千个不同的RNA分子来执行存储在染色体DNA中的指令。这些RNA分子编码并调节执行细胞功能的其他生物分子。这些RNA的合成、加工和破坏经常在空间上组织成真核生物所有领域中发现的隔室，从单细胞真菌到多细胞生物的特化细胞。这些微米大小的结构早已在不同的细胞类型和位置通过显微镜观察到，但直到最近才被确定为无膜细胞器。该项目将可视化分子接触，将与RNA转录和加工相关的无膜细胞器结合在一起。这些数据对于了解这些细胞器如何组装，它们如何发挥作用以及这些组件的形成和溶解如何调节至关重要。外展计划为当地K-12公立学校的学生提供了一个机会，为相分离蛋白质生物物理学的详细观点做出贡献。PI将1）为罗得岛高中科学推广计划开发一个模块，突出蛋白质结构和无序的生物学重要性，包括使用蛋白质折叠/相互作用视频游戏Foldit进行培训和竞赛，2）每年将该计划的两名高中生与一名本科生导师配对，进行为期一个月的蛋白质结构和相分离表征项目，3）将课程和实践培训材料存档在公共数据库中。含有大量氨基酸谷氨酰胺的非结构化/无序区域的蛋白质是体内许多无膜细胞器组装所必需的，并且足以在体外液-液相分离成蛋白质液滴。然而，在无膜细胞器内形成的接触是结构生物学中传统技术不可见的。因此，无膜细胞器的分子结构和机械功能仍然知之甚少。计划中的研究将回答一个普遍的问题：内在无序的蛋白质结构域中富含谷氨酰胺的序列如何编码与自组装和共组装成无膜细胞器相关的结构和相互作用？这个问题将通过表征富含谷氨酰胺的结构域及其组件的原子结构和相互作用，使用NMR光谱，显微镜和分子模拟来回答。在酵母和无脊椎动物中发现的三种不同的富含谷氨酰胺的蛋白质都是生理性无膜细胞器形成所必需的，它们将被用作模型。作为一个不同的蛋白质家族的代表性成员，该项目的结果将作为理解整个RNA加工组件的结构，相互作用，调控和功能的基础。将在细胞相分离和生物体表型测定中对结构模型和体外结果进行检测。这项工作是长期目标的第一步，以映射低复杂性相互作用结构域的序列，结构和复合物及其组装成无膜细胞器之间的机械联系。该项目将提供关于真核细胞生物学中这一重要但神秘现象的原子级详细信息。该奖项反映了NSF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。

Living objects carry out instructions stored in chromosomal DNA by creating thousands of distinct RNA molecules. These RNA molecules, code for, and regulate other biological molecules that perform cellular functions. Synthesis, processing, and destruction of these RNAs is frequently spatially organized into compartments found in all domains of eukaryotic life, from single-celled fungi to the specialized cells of multicellular organisms. These micron-sized structures have long been observed by microscopy in different cell types and locations but only recently have they been identified as membraneless organelles. This project will visualize the molecular contacts that hold together membraneless organelles associated with RNA transcription and processing. These data will be essential to understand how these organelles assemble, how they function, and how the formation and dissolution of these assemblies is regulated. The plan for outreach provides an opportunity for local K-12 public school students to contribute to this detailed view of phase separating protein biophysics. The PI will 1) develop a module highlighting the biological importance of protein structure and disorder for a Rhode Island high school science outreach program, including training and competition using Foldit, the protein folding/interaction video game, 2) pair two high school students per year from this program with an undergraduate student mentor for a one month project characterizing protein structure and phase separation, 3) archive the lesson and hands-on training materials in a public database. Proteins with unstructured/disordered regions containing a large amount of the amino acid glutamine are necessary for assembly of many of the membraneless organelles in vivo and are sufficient for liquid-liquid phase separation into protein droplets in vitro. Yet, the contacts formed within membraneless organelles are invisible to traditional techniques in structural biology. Therefore, membraneless organelle molecular architecture and mechanistic function remain poorly understood. The planned research will answer the general question: How do the glutamine-rich sequences in intrinsically disordered protein domains encode the structure and interactions associated with self- and co-assembly into membraneless organelles? The question will be answered by characterizing the atomic structure and interactions of glutamine-rich domains and their assemblies using NMR spectroscopy, microscopy, and molecular simulation. Three different glutamine-rich proteins found in yeast and invertebrate animals all known to be essential for physiological membraneless organelle formation will be used as models. As representative members of a diverse family of proteins, the results of the project will serve as the foundation for understanding the structure, interactions, regulation, and function of an entire class of RNA processing assemblies. Structural models and in vitro findings will be tested in established in cell phase separation and in organism phenotypic assays. This work serves as the first step in the long-term objective to map the mechanistic link between the sequence, structure and complexes of low complexity interaction domains and their assembly into membraneless organelles. This project will provide atomically detailed information on this important but mysterious phenomenon in eukaryotic cell biology.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.