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

生物体通过产生数千个不同的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。从单细胞真菌到多细胞生物的特化细胞，这些rna的合成、加工和破坏通常在空间上被组织成真核生物所有领域的隔室。这些微米大小的结构已经在显微镜下观察到不同的细胞类型和位置，但直到最近才被确定为无膜细胞器。这个项目将可视化的分子接触，保持在一起的无膜细胞器与RNA转录和加工。这些数据对于理解这些细胞器是如何组装的，它们是如何起作用的，以及这些组装的形成和溶解是如何被调节的至关重要。外展计划为当地K-12公立学校的学生提供了一个机会，为阶段分离蛋白质生物物理学的详细观点做出贡献。PI将1)为罗德岛高中科学推广项目开发一个模块，突出蛋白质结构和紊乱的生物学重要性，包括使用蛋白质折叠/互动视频游戏Foldit进行培训和比赛；2)每年将该项目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特征项目。3)在公共数据库中存档课程和实践培训材料。具有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许多无膜细胞器在体内组装所必需的，并且足以在体外液-液相分离成蛋白质滴。然而，在结构生物学的传统技术中，无膜细胞器内形成的接触是不可见的。因此，无膜细胞器的分子结构和机制功能仍然知之甚少。计划中的研究将回答一个普遍的问题：在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相关的结构和相互作用到无膜细胞器中？这个问题将通过表征谷氨酰胺富域的原子结构和相互作用，并使用核磁共振光谱，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被认为是生理无膜细胞器形成所必需的，将被用作模型。作为不同蛋白质家族的代表成员，该项目的结果将作为理解整个RNA加工组装的结构，相互作用，调节和功能的基础。结构模型和体外结果将在细胞相分离和生物表型分析中进行测试。这项工作是绘制低复杂性相互作用域的序列、结构和复合物及其组装成无膜细胞器之间的机制联系的长期目标的第一步。该项目将为真核细胞生物学中这一重要而神秘的现象提供原子细节信息。该奖项反映了美国国家科学基金会的法定使命，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.