MCA: Using Multiple Approaches for Understanding RNA Binding by Enzymes in Intermediary Metabolism

所有细胞都需要以协调的方式调整其活动以响应环境变化的方法。近年来，令人惊讶的是，数十种在从食物中获取能量中起核心作用的蛋白质被发现与特定的RNA分子有直接的相互作用，而RNA分子是调节或抑制其他细胞活动的关键分子。对这些蛋白质如何与RNA相互作用和控制RNA功能的更深入了解可能会导致设计和开发新的方法来调节多种细胞活动，以及商业生物技术应用。这个职业中期发展（MCA）项目将使首席研究员能够在国家低温电子显微镜研究设施接受确定蛋白质/RNA复合物结构的实践培训。PI还将与一位专家合作，以获得使用计算方法分析和预测蛋白质/RNA相互作用的经验。该项目将为本科生提供获得研究经验的机会，并由此获得指导和培训，为他们未来的科学职业生涯做好准备。培训具有这些技能的个人对美国的高科技产业很重要。RNA和rbp之间的动态相互作用在转录后基因调控的各个方面发挥着关键作用，包括剪接、转运、翻译、维持RNA稳定性或促进RNA降解。在一个多功能蛋白中结合催化和RNA结合功能可以成为一种协调细胞活动的机制，例如，通过酶配体的可用性来感知细胞的代谢状态，并通过调节特定转录物的翻译来做出反应。相反，RNA结合可以调节酶的催化活性。用于确定酶/RNA复合物结构的冷冻电子显微镜将补充用于分析和预测蛋白质上RNA结合位点的计算方法。结构导向诱变将用于验证RNA/蛋白质相互作用位点。该项目的成功完成将增加含有RNA与非规范RNA结合蛋白结合的复合物结构的有限数量，并提供有关RNA结合机制的宝贵信息。基于计算机的分析将有助于理解相互作用表面的性质，并将为识别其他非常规RNA结合蛋白提供基础。这些信息可以在未来应用于设计和开发调节RNA翻译，稳定性和寿命的新蛋白质以及调节酶功能的RNA。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。

All cells need methods to adjust their activities in a coordinated fashion to respond to changes in their environment. In recent years, dozens of proteins that have central roles in obtaining energy from food have, surprisingly, been found to have direct interactions with specific RNA molecules that are key players in turning up or down other cellular activities. A greater understanding of how these proteins interact with and control RNA functions may lead to the design and development of novel methods to regulate many kinds of cellular activities, as well as commercial biotechnology applications. This Mid-Career Advancement (MCA) project will enable the principal investigator to receive hands-on training in determining the structures of protein/RNA complexes at a national cryo-electron microscopy research facility. The PI will also work with an expert collaborator to gain experience in using computational methods for analysis and prediction of protein/RNA interactions. The project will generate opportunities for undergraduate students to gain research experience and with it the mentoring and training needed to prepare them for future careers in science. Training of individuals with these skills is important to the high-tech industries in the United States. Dynamic interactions between RNA and RBPs play key roles in all aspects of post-transcriptional gene regulation, including splicing, transport, translation, and maintaining RNA stability or promoting RNA degradation. Combining catalytic and RNA binding functions in one multifunctional protein can be a mechanism to coordinate cellular activities, for example, by sensing the cell’s metabolic state through availability of the enzyme’s ligands and responding by regulating translation of specific transcripts. Conversely, RNA binding could regulate the enzyme’s catalytic activity. Cryo-electron microscopy for determining the structures of enzyme/RNA complexes will complement computational methods for analysis and prediction of RNA binding sites on proteins. Structure-guided mutagenesis will be used for validation of RNA/protein interaction sites. Successful completion of the proposed project will add to the limited number of structures of complexes containing RNA bound to noncanonical RNA binding proteins and provide valuable information about the mechanisms of RNA binding. The computer-based analysis will aid in understanding the nature of the interacting surfaces and will provide a basis for identifying other unconventional RNA binding proteins. This information can be applied in the future to the design and development of novel proteins that regulate RNA translation, stability, and lifetime and RNAs that regulate enzyme function.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.