Deciphering the Code for RNA Recognition by PPR Proteins

智力优势：细胞内的分子机制对遗传信息的解释需要蛋白质、DNA和RNA分子之间高度特异性的相互作用。预测RNA和DNA结合蛋白结合的核苷酸序列的能力将加速对生物体发育和生理学基础的分子网络的理解，并将在基础研究和生物技术中具有广泛的应用。然而，仍然难以预测大多数RNA结合蛋白的RNA结合特性，或者设计它们以结合特定的RNA序列。该项目的重点是一个家庭的RNA结合蛋白，五肽重复（PPR）蛋白，持有特殊的承诺，为合理设计的特定RNA结合特性和工程的调控开关，以控制基因表达。PPR蛋白由形成结合RNA的表面的串联重复单元组成。PPR-RNA识别本质上是模块化的：每个重复序列通过重复序列中两个位置处氨基酸的组合作用指定与特定RNA核苷酸的结合。PPR蛋白存在于所有真核生物中，但该家族在植物谱系中的规模非常大，在开花植物中有450个成员。PPR蛋白主要定位于线粒体和叶绿体，在那里它们影响由细胞器基因组编码的RNA的表达。因此，PPR家族对许多细胞器依赖性过程具有深远的影响，包括光合作用，呼吸和生育力。该项目的目标是发展对PPR-RNA识别基础的理解，以便（i）可以可靠地预测作物基因组中PPR蛋白的结合位点和功能，以及（ii）可以设计PPR蛋白以实现所需的RNA序列特异性。预测PPR结合位点和该结合的功能后果的能力将通过工程化PPR蛋白来调节与其天然靶不同的内源性细胞器基因来测试。更广泛的影响：预期的结果将通过以下方式影响应用和基础生物学：（i）加速植物中最大的基因家族之一的功能分配;（ii）提供一组控制线粒体和叶绿体基因表达的开关，从而调节光合作用，呼吸作用和植物生育力;（iii）提供将乘客蛋白质靶向体内特定RNA序列的工具。这笔资金将促进美国和澳大利亚实验室之间的国际合作。这项研究的跨学科和国际性质将为一名研究生和两名本科生提供丰富的教育经验。本科生将拥有该项目的核心组成部分，并将采用信息学，生物化学和遗传学方法。他们将由PI和研究生在一个项目中指导，该项目将包括根据他们的需求量身定制的每周小组会议。项目内容也将被纳入旨在增加STEM学科多样性的全校性举措。该项目由分子和细胞生物科学部的遗传机制、综合有机系统部的植物基因组研究计划和国际科学与工程办公室共同资助。

Intellectual Merit: The interpretation of genetic information by the molecular machinery inside cells requires highly specific interactions between protein, DNA, and RNA molecules. The ability to predict the nucleotide sequences bound by RNA and DNA binding proteins would accelerate understanding of the molecular networks that underlie organismal development and physiology, and would have broad application in basic research and biotechnology. However, it remains difficult to predict the RNA binding properties of most RNA binding proteins, or to design them to bind specified RNA sequences. This project focuses on a family of RNA binding proteins, the pentatricopeptide repeat (PPR) proteins, that holds special promise for the rational design of specified RNA binding properties and for the engineering of regulatory switches to control gene expression. PPR proteins consist of tandem repeating units that form a surface for binding RNA. PPR-RNA recognition is modular in nature: each repeat specifies binding to a particular RNA nucleotide via the combinatorial action of amino acids at two positions in the repeat. PPR proteins are found in all eucaryotic organisms, but the family is extraordinary for its size in the plant lineage, with 450 members in flowering plants. PPR proteins localize primarily to mitochondria and chloroplasts, where they influence the expression of RNAs encoded by the organellar genomes. As such, the PPR family has a profound impact on many organelle-dependent processes, including photosynthesis, respiration, and fertility. The goal of this project is to develop understanding of the basis for PPR-RNA recognition to the point that (i) the binding sites and functions of PPR proteins in crop plant genomes can be reliably predicted, and (ii) PPR proteins can be designed to achieve desired RNA sequence specificities. The ability to predict PPR binding sites and the functional consequences of that binding will be tested by engineering PPR proteins to regulate endogenous organellar genes that are distinct from their natural targets. Broader Impacts: Anticipated outcomes would impact applied and basic biology by: (i) accelerating the assignment of functions to one of the largest gene families in plants; (ii) providing a set of switches with which to control mitochondrial and chloroplast gene expression, and thereby modulate photosynthesis, respiration, and plant fertility; and (iii) providing tools for targeting passenger proteins to specified RNA sequences in vivo. This funding will foster an international collaboration between laboratories in the US and Australia. The interdisciplinary and international nature of this research will provide a rich educational experience for one graduate and two undergraduate students. The undergraduates will take ownership of core components of the project, and will employ informatic, biochemical and genetic approaches. They will be mentored by the PI and graduate student in a program that will include weekly subgroup meetings tailored to their needs. Project content will also be incorporated into University-wide initiatives aiming to increase diversity in STEM disciplines. This project is co-funded by Genetic Mechanisms in the Division of Molecular and Cellular Biosciences, by Plant Genome Research Program in the Division of Integrative Organismal Systems, and by the Office of International Science and Engineering.