Deciphering the Code for RNA Recognition by PPR Proteins

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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.