EAGER: Exploring the potential of pentatricopeptide repeat proteins for the site-directed modulation of RNA metabolism

该奖项是根据2009年美国复苏和再投资法案(公法111-5)资助的。智力价值通过“设计者”调控元件调节基因表达的具体步骤的能力在基础研究和生物技术中有着广泛的应用。RNA结合蛋白可以调节RNA介导的事件，但由于很难根据其序列或结构来预测其RNA结合特性，因此大多数类型的RNA结合蛋白都不适合工程应用。在此背景下，PUM-HD的不同寻常的RNA识别机制引起了人们的极大关注：PUM-HD由8个螺旋重复组成，通过一重复、一核苷酸的机制识别连续的8-9个核苷酸片段。这种模块化的识别机制为PUM-HD识别RNA提供了一种“代码”，并为设计PUM-HD以结合新的RNA靶标提供了可能性。该项目专注于一类不太知名的蛋白质，五肽重复(PPR)蛋白质，它为设计新的RNA结合特异性和设计基因调控系统提供了更大的希望。预计PPR蛋白将采用螺旋重复螺线管结构，这让人想起PUM-HD。目前的数据表明，PPR区域像PUM-HD一样，通过一种模块化的识别机制与RNA结合，其规则应该是可定义的。然而，PPR蛋白中高度可变的重复数及其天然RNA配体和生理功能的显著多样性支持这样的观点，即PPR束为RNA结合提供了一个更具延展性的平台。此外，PPR蛋白沿着异常长的界面结合单链RNA的能力赋予了一系列不同寻常的生化活性，当PPR蛋白被靶向特定位置时，这些活性可以预测对基因表达的特定影响。该项目将(I)测试PPR蛋白的不同寻常的特性可以被用来在不同的生物体中以不同的步骤调节基因表达的前提，以及(Ii)在理解PPR蛋白与序列特定的RNA结合的“密码”方面取得实质性进展。广泛影响将PPR基序开发为工程应用的平台将需要设计PPR蛋白来识别各种RNA的能力，以及关于可以由PPR/RNA相互作用调节的基因表达步骤的知识。这个项目解决了这两个问题，因此将为开发新的工具来操纵原核生物和真核生物的基因表达奠定基础。成功的结果将为设计功能强大的选择方案提供基础，用于PPR蛋白质的定向进化以识别新的RNA配体。此外，这些结果将提供对这个庞大而鲜为人知的RNA结合蛋白家族的机械性见解，它在所有真核生物的细胞器基因表达(从而在能量转导)中扮演着许多重要的角色。这项研究的跨学科性质以及所采用的生物和化验方法的多样性将为本科生提供丰富的教育经验。同一名学生将参与整个为期两年的项目，这样学生就可以拥有一个目标，并从头到尾坚持下去。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Intellectual MeritThe ability to modulate specific steps in gene expression via "designer" regulatory elements has broad application in basic research and biotechnology. RNA binding proteins can modulate RNA-mediated events, but most classes of RNA binding protein are poor candidates for engineering applications due to the difficulty of predicting their RNA binding properties from their sequence or structure. In this context, the unusual RNA recognition mechanism of the Pumilio Homology Domain (PUM-HD) has attracted considerable attention: the PUM-HD consists of eight helical repeats that recognize a contiguous 8-9 nucleotide RNA segment via a one-repeat, one-nucleotide mechanism. This modular recognition mechanism suggested a "code" for RNA recognition by the PUM-HD, and offers the possibility of engineering the PUM-HD to bind novel RNA targets. This project focuses on a less well-known protein class, the pentatricopeptide repeat (PPR) proteins, which offer greater promise for the design of novel RNA binding specificities and for the engineering of gene regulatory systems. PPR proteins are predicted to adopt a helical repeat solenoid structure that is reminiscent of the PUM-HD. Current data suggest that PPR tracts, like the PUM-HD, bind RNA via a modular recognition mechanism whose rules should be definable. However, the highly variable number of repeats in PPR proteins and the remarkable diversity of their natural RNA ligands and physiological functions support the view that PPR tracts provide a much more malleable platform for RNA binding. Furthermore, the ability of PPR proteins to bind single-stranded RNA along an unusually long interface imparts an unusual repertoire of biochemical activities, which predict specific effects on gene expression when PPR proteins are targeted to specific sites. This project will (i) test the premise that the unusual properties of PPR proteins can be exploited to modulate gene expression at diverse steps in diverse organisms, and (ii) make substantial progress towards understanding the "code" for sequence-specific RNA binding by PPR proteins.Broader ImpactsDevelopment of the PPR motif as a platform for engineering applications will require the ability to design PPR proteins to recognize a wide variety of RNAs, and knowledge about the steps in gene expression that can be modulated by PPR/RNA interactions. This project addresses both of these issues, and will thereby provide the foundation for the development of new tools for the manipulation of gene expression in both prokaryotes and eukaryotes. Successful outcomes will provide the basis for the design of powerful selection schemes for the directed evolution of PPR proteins to recognize new RNA ligands. Furthermore, the results will provide mechanistic insights into this large and poorly-understood family of RNA binding proteins, which plays numerous essential roles in organellar gene expression (and thus in energy transduction) in all eukaryotes. The interdisciplinary nature of this research and the diversity of the organisms and assays to be employed will provide a rich educational experience for an undergraduate student. The same student will participate throughout the two-year project, such that the student can take ownership of one aim and follow it through from beginning to end.