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的方法，以应对环境的变化。 近年来，令人惊讶的是，数十种在从食物中获得能量方面发挥核心作用的蛋白质被发现与特定的RNA分子直接相互作用，这些RNA分子是增加或减少其他细胞活动的关键因素。更好地了解这些蛋白质如何与RNA相互作用并控制RNA功能，可能会导致设计和开发调节多种细胞活动的新方法，以及商业生物技术应用。这个中期职业发展（MCA）项目将使主要研究者能够在国家冷冻电子显微镜研究设施中接受确定蛋白质/RNA复合物结构的实践培训。PI还将与专家合作，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，并随之提供为他们未来的科学职业做好准备所需的指导和培训。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关键作用，包括剪接、转运、翻译和维持RNA稳定性或促进RNA降解。在一个多功能蛋白质中组合催化和RNA结合功能可以是协调细胞活性的机制，例如，通过酶的配体的可用性来感测细胞的代谢状态，并通过调节特定转录物的翻译来响应。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。将使用结构指导诱变验证RNA/蛋白质相互作用位点。该项目的成功完成将增加有限数量的含有与非典型RNA结合蛋白结合的RNA的复合物的结构，并提供有关RNA结合机制的有价值的信息。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用于设计和开发调节RNA翻译、稳定性和寿命的新型蛋白质以及调节酶功能的RNA。该奖项反映了NSF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.