Multi-scale computational investigation of functions and mechanisms of protein-RNA phase separation.

项目总结/摘要 近年来，已经出现了一个惊人的认识，即蛋白质和核酸的液-液相分离 酸负责各种细胞内无膜细胞器的形成。细胞器的例子 相分离形成的是细胞核中的核仁和Cajal小体，细胞核中的应力颗粒和P颗粒。 细胞质蛋白质-RNA复合物的相分离，特别是，被认为是至关重要的 基因调控过程与真核生物表型复杂性的联系。尽管外观- 我们的生物学意义和广泛的实验努力，我们对联系机制的理解， 仍然缺乏具有转录和催化过程的蛋白质-RNA相分离。根本 （i）RNA和蛋白质的分子异质性和构象不稳定性， 包含低复杂性无序区域（ii）分子和细胞尺度的并列（iii） 由于生化反应、ATP驱动的过程和不可逆的生物聚合物絮凝引起的非平衡效应。 目前的理论和计算范式往往缺乏最佳的时空分辨率和正确的 结合物理见解，以全面和综合的方式面对复杂的实验数据。 方式在这里，我建议使用我们实验室开发的多尺度计算工具， 揭示蛋白质-RNA相分离及其联系的一般机械原理的数据驱动方法 与功能性的监管过程。该建议包括三个方向。在第一个方向，我们专注于 蛋白质和RNA的分级粗粒化研究液-液热力学驱动力 通过分子动力学技术在体外进行相分离。在第二个方向上，我们使用有限元素 以及由分子模型和实验数据训练的反应扩散模拟， 具有转录和催化反应的液-液相分离，这是体内 条件在第三个方向，我们评估了蛋白质-RNA相分离对基因调控的影响。 网络使用随机动力学模拟。为研究选择的特定系统是实验性的- 完全表征的RNA结合蛋白FUS、TDP-43、Tau、hnrpa 1和hnrpa 2。这些系统是已知的 用于在通常调节的条件下形成液体蛋白质-RNA缩合物和聚集结构， 失调，从而导致严重的神经退行性疾病。 拟议的研究计划的完成将阐明蛋白质-RNA阶段分离的性质， 定量其与功能性生化反应的联系，并为制定干预措施提供急需的见解 阻止蛋白质聚集到诱导细胞体的疾病中的策略。

PROJECT SUMMARY/ABSTRACT In recent years there has emerged a striking realization that liquid-liquid phase separation of proteins and nucleic acids is responsible for the formation of various intracellular membraneless organelles. Examples of organelles formed by phase separation are nucleoli and Cajal bodies in the nucleus and stress granules and P granules in the cytoplasm. The phase separation of protein-RNA composites, in particular, is being appreciated for crucial roles of connecting gene regulatory processes with the phenotypic complexity of eukaryotes. Despite the appar- ent biological significance and extensive experimental efforts, our understanding of the mechanisms which link protein-RNA phase separation with the transcriptional and catalytic processes is still lacking. The fundamental challenges stem from (i) The molecular heterogeneity and conformational flexibility of RNA and proteins, which contain low complexity disordered regions (ii) The juxtaposition of molecular and cellular scales (iii) Presence of non-equilibrium effects due to biochemical reactions, ATP driven processes, and irreversible bio-polymer fluxes. The current theoretical and computational paradigms often lack optimal spatio-temporal resolution and the right combination of physical insights for confronting the complex experimental data in a comprehensive and integrative manner. Here, I propose using multi-scale computational tools developed in our lab combined in conjunction with data-driven approaches for revealing general mechanistic principles of protein-RNA phase separation and its link with the functional regulatory processes. The proposal consists of three directions. In the first direction, we focus on hierarchical coarse-graining of proteins and RNA for studying thermodynamic driving forces of liquid-liquid phase separation in in vitro via molecular dynamics techniques. In the second direction, we employ finite-element and reaction-diffusion simulations trained by molecular models and experimental data for studying the connec- tion of liquid-liquid phase separation with transcriptional and catalytic reactions, which is characteristic of in vivo conditions. In the third direction, we assess the impact of protein-RNA phase separation generic gene regulatory networks by using stochastic dynamics simulations. The specific systems chosen for the study are experimen- tally well-characterized RNA binding proteins FUS, TDP-43, Tau, hnrpa1, and hnrpa2. These systems are known for forming liquid protein-RNA condensates under usually regulated conditions and aggregated structures when misregulated, thereby leading to major neurodegenerative diseases. The completion of the proposed research program will elucidate the nature of the protein-RNA phase sepa- ration its link with functional biochemical reactions and provide much-needed insights for developing intervention strategies for halting protein aggregation into diseases inducing cellular bodies.