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。智力优势通过“设计师”调控元件调节基因表达中特定步骤的能力在基础研究和生物技术中有广泛的应用。RNA结合蛋白可以调节RNA介导的事件，但是大多数种类的RNA结合蛋白由于难以从它们的序列或结构预测它们的RNA结合性质而对于工程应用是差的候选者。在这种情况下，Pumilio同源结构域（Pumilio Homology Domain，Pumilio-HD）的不寻常的RNA识别机制引起了相当大的关注：Pumilio-HD由八个螺旋重复序列组成，通过一个重复序列，一个核苷酸的机制识别连续的8-9个核苷酸的RNA片段。这种模块化的识别机制表明，一个“代码”的RNA识别的BLO-HD，并提供了工程的BLO-HD结合新的RNA靶点的可能性。该项目的重点是一个不太知名的蛋白质类，五肽重复（PPR）蛋白质，这提供了更大的承诺，设计新的RNA结合特异性和基因调控系统的工程。PPR蛋白被预测为采用螺旋重复螺线管结构，这让人联想到的EST-HD。目前的数据表明，PPR tracts，如EST-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，在PPR蛋白的重复序列的高度可变的数量和显着的多样性，其天然RNA配体和生理功能支持的观点，PPR束提供了一个更具可塑性的平台，RNA结合。此外，PPR蛋白质结合单链RNA的能力沿着一个异常长的界面赋予了一个不寻常的库的生化活动，预测特定的影响基因表达时，PPR蛋白质靶向特定的网站。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体中不同步骤的基因表达的前提，以及（ii）在理解序列的“代码”方面取得实质性进展-PPR基序作为工程应用平台的发展将需要设计PPR蛋白的能力，以识别各种各样的以及关于可以通过PPR/RNA相互作用调节的基因表达步骤的知识。该项目解决了这两个问题，从而将为原核生物和真核生物中基因表达操纵的新工具的开发提供基础。成功的结果将为设计强有力的选择方案提供基础，用于PPR蛋白的定向进化以识别新的RNA配体。此外，这些结果将提供机制的见解，这个大的和不太了解的RNA结合蛋白家族，在细胞器基因表达（从而在能量转导）在所有真核生物中发挥着许多重要作用。这项研究的跨学科性质和生物体和测定的多样性将为本科生提供丰富的教育经验。同一个学生将参与整个两年的项目，这样学生就可以拥有一个目标，并从开始到结束。

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.