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.

As part of our project, we successfully designed new tools based on synthetic proteins, called synPPR, capable of stabilizing target mRNAs in plant chloroplasts and precisely editing RNA bases in both chloroplasts and mitochondria. These tools enable the rewriting of genetic information in mRNAs and allow targeted control of gene expression.

However, the use of synPPR to stabilize mRNAs in the cytoplasm of bacteria or the nucleus of human cells did not achieve the expected success, likely due to the greater complexity of cytonuclear transcriptomes compared to those of plant organelles.

The project has opened several promising perspectives, both scientifically and technologically. First, the development of synPPR tools capable of modulating RNA expression in chloroplasts and mitochondria paves the way for applications in plant biotechnology, particularly for improving agronomic traits. By enabling targeted RNA editing within organelles, these technologies could contribute to the creation of new plant varieties with improved resistance to environmental stresses, enhanced photosynthetic efficiency, or specific characteristics such as cytoplasmic male sterility (CMS) and fertility restoration for hybrid seed production.

On a fundamental level, these synPPR tools also offer opportunities to better understand post-transcriptional regulation mechanisms and RNA-protein interactions within plant organelles. The ability to precisely target RNAs could potentially be extended to other biological systems, opening new avenues for research in human genetics, particularly in the context of mitochondrial diseases.

Finally, the technical challenges encountered, particularly regarding the expression of synPPRs and the control of their RNA-binding specificity, highlight the need for continued research efforts to optimize these tools and expand their applications beyond plant organelles. These developments could also lead to advances in industrial biotechnology, where fine regulation of gene expression is crucial.

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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.