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。通过“设计者”调节元件来调节基因表达的特定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，但由于难以从其序列或结构预测其RNA结合特性，大多数类型的RNA结合蛋白不适合用于工程应用。在此背景下，Pumilio同源结构域（pumi - hd）不同寻常的RNA识别机制引起了相当大的关注：pumi - hd由8个螺旋重复组成，通过一个重复、一个核苷酸的机制识别连续的8-9个核苷酸RNA片段。这种模块化识别机制提示了puma - hd识别RNA的“代码”，并提供了设计puma - hd结合新RNA靶标的可能性。该项目重点研究一类不太为人所知的蛋白质，即五肽重复（PPR）蛋白质，它为设计新的RNA结合特异性和基因调控系统的工程提供了更大的希望。预测PPR蛋白采用螺旋重复螺线管结构，这让人想起puma - hd。目前的数据表明，小反刍病毒束，如puma - hd，通过模块化识别机制结合RNA，其规则应该是可定义的。然而，PPR蛋白中高度可变的重复次数及其天然RNA配体和生理功能的显著多样性支持了PPR束为RNA结合提供了更具可塑性的平台的观点。此外，PPR蛋白沿着异常长的界面结合单链RNA的能力赋予了不寻常的生化活性库，这可以预测当PPR蛋白靶向特定位点时对基因表达的特定影响。该项目将(i)测试PPR蛋白的不寻常特性可以被利用来调节不同生物中不同步骤的基因表达的前提，以及（ii）在理解PPR蛋白序列特异性RNA结合的“代码”方面取得实质性进展。更广泛的影响PPR基序作为工程应用平台的发展将需要设计PPR蛋白以识别各种RNA的能力，以及关于PPR/RNA相互作用可以调节的基因表达步骤的知识。该项目解决了这两个问题，并将因此为开发用于操纵原核生物和真核生物基因表达的新工具提供基础。成功的结果将为PPR蛋白定向进化识别新的RNA配体的强大选择方案的设计提供基础。此外，这些结果将为这个庞大而知之甚少的RNA结合蛋白家族提供机制见解，该家族在所有真核生物的细胞器基因表达（以及能量转导）中起着许多重要作用。本研究的跨学科性质以及所采用的生物和测定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，并从头到尾跟随它。

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.