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

职业：转录和 RNA 加工中富含谷氨酰胺域液-液相分离的结构方面

• 批准号: 1845734
• 负责人: Nicolas Fawzi
• 金额: $ 91.37万
• 依托单位: Brown University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1845734&HistoricalAwards=false
• 关键词: CAREER; Structural; aspects; glutamine; rich

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.

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。这些 RNA 的合成、加工和破坏通常在空间上组织成真核生命所有领域（从单细胞真菌到多细胞生物的特殊细胞）中发现的区室。长期以来，人们一直通过显微镜在不同的细胞类型和位置观察到这些微米大小的结构，但直到最近才将它们鉴定为无膜细胞器。该项目将可视化将与 RNA 转录和加工相关的无膜细胞器结合在一起的分子接触。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如何调节这些组装体的形成和溶解至关重要。该推广计划为当地 K-12 公立学校学生提供了一个机会，让他们为相分离蛋白质生物物理学的详细观点做出贡献。 PI 将 1) 为罗德岛州高中科学推广计划开发一个模块，强调蛋白质结构和无序的生物学重要性，包括使用 Foldit（蛋白质折叠/互动视频游戏）进行训练和竞赛，2) 每年将本项目的两名高中生与一名本科生导师配对，进行为期一个月的表征蛋白质结构和相分离的项目，3) 将课程和实践培训材料存档在公共数据库中。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体内组装许多无膜细胞器所必需的，并且足以在体外液-液相分离成蛋白质液滴。然而，无膜细胞器内形成的接触对于结构生物学的传统技术来说是不可见的。因此，对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这个普遍问题：本质上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？通过使用核磁共振波谱、显微镜和分子模拟表征富含谷氨酰胺结构域及其组装的原子结构和相互作用，可以回答这个问题。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将被用作模型，它们都已知对生理无膜细胞器的形成至关重要。作为不同蛋白质家族的代表性成员，该项目的结果将成为理解整类 RNA 加工组件的结构、相互作用、调节和功能的基础。结构模型和体外研究结果将在细胞相分离和生物体表型测定中进行测试。这项工作是绘制低复杂性相互作用域的序列、结构和复合物之间的机制联系及其组装成无膜细胞器的长期目标的第一步。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智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Interactions between ALS-linked FUS and nucleoporins are associated with defects in the nucleocytoplasmic transport pathway.

• DOI: 10.1038/s41593-021-00859-9
• 发表时间: 2021-08
• 期刊: Nature neuroscience
• 影响因子: 25
• 作者: Lin YC;Kumar MS;Ramesh N;Anderson EN;Nguyen AT;Kim B;Cheung S;McDonough JA;Skarnes WC;Lopez-Gonzalez R;Landers JE;Fawzi NL;Mackenzie IRA;Lee EB;Nickerson JA;Grunwald D;Pandey UB;Bosco DA
• 通讯作者: Bosco DA

A predictive coarse-grained model for position-specific effects of post-translational modifications

• DOI: 10.1016/j.bpj.2021.01.034
• 发表时间: 2021-04-06
• 期刊: BIOPHYSICAL JOURNAL
• 影响因子: 3.4
• 作者: Perdikari, Theodora Myrto;Jovic, Nina;Mittal, Jeetain
• 通讯作者: Mittal, Jeetain

Epigenetic cell fate in Candida albicans is controlled by transcription factor condensates acting at super-enhancer-like elements.

• DOI: 10.1038/s41564-020-0760-7
• 发表时间: 2020-11
• 期刊: Nature microbiology
• 影响因子: 28.3
• 作者: Frazer C;Staples MI;Kim Y;Hirakawa M;Dowell MA;Johnson NV;Hernday AD;Ryan VH;Fawzi NL;Finkelstein IJ;Bennett RJ
• 通讯作者: Bennett RJ

Membrane bending by protein phase separation.

通过蛋白质相分离使膜弯曲。

• DOI: 10.1073/pnas.2017435118
• 发表时间: 2021
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Yuan,Feng;Alimohamadi,Haleh;Bakka,Brandon;Trementozzi,AndreaN;Day,KaseyJ;Fawzi,NicolasL;Rangamani,Padmini;Stachowiak,JeanneC
• 通讯作者: Stachowiak,JeanneC