CE20 - Biologie des animaux, des organismes photosynthétiques et des microorganismes

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

Submission summary

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.

Project coordinator

CNRS-Institut de biologie moléculaire des plantes (IBMP) (Laboratoire public)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

Centre de Recherche en Myologie
INRA Institut Jean Pierre Bourgin
CNRS-Institut de biologie moléculaire des plantes (IBMP)

Help of the ANR 548,365 euros
Beginning and duration of the scientific project: December 2018 - 48 Months

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