Engineering synthetic PPR motif RNA binding proteins for the site-specific regulation of gene expression in living organisms – SynPPR

PPR proteins bind RNA according to a simple and specific recognition amino acid combinatorial code that allows to custom-design proteins that bind any desired RNA sequence. In this project, we will use the PPR code to engineer and program proteins to bind desired RNA sequences. We use transgenesis to express them in various organisms to target specific mRNAs and control their fate and therefore, the expression of their genes.

We demonstrated that it was possible to use custome-made synthetic PPR repeat proteins to control the expression of specific genes in chloroplasts by the regulation of their mRNA stability in vivo.

These results open the door to the development of many applications for synthetic PPR proteins in living organisms. We will try to express synthetic PPRs in mitochondria of plants and in bacteria and the nucleus of human cells to regulate the expression of their genes.

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The manipulation of gene expression represents a major challenge for both, the basic and applied research. However, existing methods are increasingly focused on the nuclear genome that contains almost all genes found in eukaryotic cells and often, cannot be easily applied to mitochondria or chloroplasts, two essential energetic organelles that contain their own genomes. The crucial role of post-transcriptional events in determining the outcome of gene expression and the recognized implication of RNA molecules in severe diseases in humans or in plant fertility have motivated the development of synthetic biology approaches that target RNA and modulate gene functions. RNA binding proteins play essential roles in every aspect of RNA metabolism and the ability to engineer such proteins to bind a specified RNA sequence would provide new avenues for the genetic engineering. However, most common RNA binding motifs are poor candidates for protein engineering. In this context, Pentatricopeptide repeat (PPR) proteins are an attractive class of RNA binding proteins whose modular helical repeat architecture and RNA binding mechanism offer an ideal platform for the engineering of programmable RNA binders. PPR proteins are nuclear encoded and function in the posttranscriptional regulation of organellar gene expression. PPR proteins are made of a variable number of a degenerate 35-amino acid repeat that bind specific RNA sequences following a mechanism in which each repeat recognizes a single RNA base through a combinatorial 2-amino acid code. This flexible repeat architecture and the binding code enable the rational design of PPR proteins with desired RNA binding specificity. Exploiting an optimized synthetic PPR scaffold and the PPR code, we have demonstrated that synthetic PPR repeats (SynPPRs) can be rationally programmed to bind any RNA sequence, opening the door to numerous biotechnological applications when customized SynPPRs are targeted to specific RNA-sites in vivo.
The SynPPR project overarching goal is to implement PPR-tools for useful applications in plant biology, agronomy and biomedicines, and is organized into 3 work-packages (WP). This project will constitute an iterative process with feed-back loops between its different parts. In WP1, we will exploit SynPPR designers to regulate the expression of plant organellar genes in Arabidopsis and control pollen production in crops. In a second ground-breaking WP2, we will design SynPPRs to repress toxic RNAs in the nucleus of human diseased cells. Finally, in the last WP3, we will engineer an innovative high-throughput bacterial screening assay to select protein-RNA interactions and strengthen our current understanding of PPR-RNA binding mechanism. These results will be used to refine the design of SynPPR design in WP1-2.
This interdisciplinary project will take advantage of a synergetic consortium of 3 teams with complementary expertise at both scientific and technical levels, which are at the forefront of research in their respective area: PPR protein biochemistry and functions (K. Hammani: CNRS-IBMP), mitochondria control of pollen production in plant mitochondrial translation (H. Mireau: INRA-IJPB) and the development of biotherapies for myotonic dystrophy (D. Furling: INSERM-CRM).
The results and knowledge gained from this project will benefit to both applied and basic research and will have strong economical and scientific impacts by delivering, (1) a new method for controlling of organellar gene expression in plants, (2) a new way to produce optimal male fertility restorer genes and thereby facilitate hybrid seed production in plants, (3) a tool for the manipulation of RNA in human cells and therapeutics for human RNA-dominant diseases and finally, (4) an innovative high-throughput assay for the direct selection of protein and RNA interactions in bacteria.