CAREER: Molecular mechanisms of ribonucleoprotein assembly

核糖核酸(RNA)是一种重要的生物分子，在细胞过程中发挥着不同的作用。为了实现其功能，RNA必须首先折叠成正确的结构，并与所需的辅因子(如蛋白质)组装。尽管在生物学中起着核心作用，但人们对RNA如何折叠并与蛋白质结合缺乏全面的了解。为了解决这些知识上的差距，该项目使用了7SK核糖核蛋白(RNP)复合体作为模型系统，RNP是细胞功能的关键调节因子。此外，该项目将表征合成RNA的折叠，这些RNA结合染料成为荧光，当标记到具有生物重要性的RNA上时，可以用来观察活细胞中的RNA。该项目的结果将为生物过程中涉及的RNA的RNA结构-功能关系提供基本的见解，并产生RNP组装的一般规则。该项目将教育与研究结合在一起，以增加下一代科学家的留存和多样性。该项目将开展教育活动，包括将基于发现的化学生物学实验室课程纳入本科课程。将与内布拉斯加州大学林肯分校现有的项目合作举办动手研讨会，让公众参与RNA科学。RNP对细胞功能是必不可少的，但如果没有对RNA结构和RNP组装的全面了解，指导细胞功能的分子机制就不能完全理解。7SK RNP是真核生物转录的主要调控因子，是促进对RNA调控的细胞功能有限认识的理想模型系统。对7SK RNP结构和组装的基本了解将促进对其他调控RNP转录调控和组装的理解；然而，在理解7SK RNA如何折叠和组装成功能RNP方面存在重大差距。为了克服这些差距，该项目采用多学科方法来确定7SK RNA的结构动力学，表征对调节功能至关重要的7SK RNA与蛋白质的相互作用，并将荧光RNA适配子整合到7SK RNA中用于活体细胞成像。为了实现这些目标，将综合使用溶液核磁共振光谱学、X射线结晶学、细胞方法、结合分析和荧光显微镜方法。该项目将结合促进和支持STEM中代表性不足的社区的教育活动，利用综合生物物理和化学生物学方法阐明RNA折叠和RNP组装的分子机制。该项目由生物科学局分子和细胞生物科学部的分子生物物理学分部和已建立的激励竞争研究计划(EPSCoR)共同支持。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Ribonucleic acid (RNA) is an essential biological molecule that performs diverse roles in cellular processes. To carry out its function, RNA must first fold into the correct structure and assemble with required cofactors such as proteins. Despite having a central role in biology, a comprehensive understanding of how RNA folds and binds to protein is lacking. To address these gaps in knowledge, this project uses the 7SK ribonucleoprotein (RNP) complex, a crucial regulator of cell function, as a model system. In addition, this project will characterize folding of synthetic RNAs that bind dyes to become fluorescent that, when tagged onto biologically important RNAs, can be used to observe RNA in live cells. Results arising from the project will provide fundamental insights into RNA structure-function relationships for RNAs involved in biological processes and produce general rules for RNP assembly. The project entwines education alongside research to increase retention and diversity of the next generation of scientists. This project will develop educational activities including incorporating discovery-based chemical biology laboratory coursework into the undergraduate curriculum. Hands-on workshops will be held in partnership with established programs at the University of Nebraska – Lincoln to engage the public in RNA science. RNPs are essential for cell function, yet without comprehensive knowledge of RNA structure and RNP assembly the molecular mechanisms directing cell function cannot be fully understood. The 7SK RNP, a major regulator of eukaryotic transcription, is an ideal model system to advance limited knowledge of RNA-regulated cell function. A fundamental understanding of 7SK RNP structure and assembly will advance understanding of transcription regulation and assembly of other regulatory RNPs; however, there are significant gaps in understanding how 7SK RNA folds and assembles into a functional RNP. To overcome these gaps, this project employs multidisciplinary approaches to determine 7SK RNA structural dynamics, characterize 7SK RNA-protein interactions important for regulatory function; and integrate a fluorescent RNA aptamer into 7SK RNA for live cell imaging in vivo. To achieve these objectives, combined solution NMR spectroscopy, X-ray crystallography, cellular approaches, binding assays, and fluorescence microscopy methods will be used. This project will elucidate the molecular mechanisms of RNA folding and RNP assembly using integrated biophysical and chemical biology approaches, in concert with education activities to advance and support underrepresented communities in STEM. This project is jointly supported by the Molecular Biophysics Cluster of the Molecular and Cellular Biosciences Division in the Directorate for Biological Sciences and the Established Program to Stimulate Competitive Research (EPSCoR).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.