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

基本信息

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

项目摘要

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.

活物体通过创建数千种不同的RNA分子来执行存储在染色体DNA中的说明。这些RNA分子，为执行细胞功能的其他生物分子编码和调节。这些RNA的合成，加工和破坏经常被空间组织成在真核生命的所有结构域中，从单细胞真菌到多细胞生物的专用细胞。这些微米大小的结构长期以来通过显微镜在不同的细胞类型和位置观察到，但直到最近才被鉴定为膜细胞器。该项目将可视化与RNA转录和加工相关的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，它们的功能以及如何调节这些组件的形成和溶解至关重要。外展计划为当地的K-12公立学校学生提供了机会，为分离蛋白质生物物理学的相位详细观点做出了贡献。 PI将1）开发一个模块，该模块突出了罗德岛高中科学外展计划的蛋白质结构和混乱的生物学重要性，包括使用折叠的培训和竞争，蛋白质折叠/互动视频游戏，2）每年从该计划中配对两个高中生，与一个月的学生导师一起进行一个月的蛋白质结构和相位分离的培训，3）培训材料，培训材料，培训材料和培训材料。具有大量氨基酸谷氨酰胺的非结构化/无序区域的蛋白质对于在体内组装许多无膜细胞器是必需的，并且足以使液 - 液相分离为体外蛋白液滴。然而，在结构生物学中，传统技术在膜细胞器中形成的接触是不可见的。因此，无膜细胞器分子结构和机械功能仍然了解不足。计划的研究将回答一个总体问题：本质上无序蛋白质结构域中富含谷氨酰胺的序列如何编码与自我和共组合相关的结构和相互作用，以形成膜无膜细胞器？通过表征NMR光谱，显微镜和分子模拟的原子结构以及它们的组件的相互作用，将回答这个问题。在酵母和无脊椎动物动物中发现的三种不同的富含谷氨酰胺的蛋白质都将用作生理膜细胞器形成必不可少的蛋白质。作为多元化蛋白质家族的代表成员，该项目的结果将是理解整个RNA处理组件的结构，相互作用，调节和功能的基础。结构模型和体外发现将在细胞相分离和有机体表型测定中进行测试。这项工作是长期目标的第一步，即绘制低复杂性相互作用域的序列，结构和复合物之间的机械联系，并将其组装到膜细胞器中。该项目将在真核细胞生物学中提供有关这种重要但神秘的现象的原子详细信息。该奖项反映了NSF的法定任务，并使用基金会的知识分子优点和更广泛的影响审查标准，被认为值得通过评估来提供支持。

项目成果

期刊论文数量（14）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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

The (un)structural biology of biomolecular liquid-liquid phase separation using NMR spectroscopy

DOI：
10.1074/jbc.rev119.009847
发表时间：
2020-02-21
期刊：
JOURNAL OF BIOLOGICAL CHEMISTRY
影响因子：
4.8
作者：
Murthy, Anastasia C.;Fawzi, Nicolas L.
通讯作者：
Fawzi, Nicolas L.

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