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Engineering synthetic pentatricopeptide repeat proteins for the site-specific genetic manipulation of plant organelles

Periodic Reporting for period 1 - PPRdesign (Engineering synthetic pentatricopeptide repeat proteins for the site-specific genetic manipulation of plant organelles )

Reporting period: 2018-06-01 to 2020-05-31

Mitochondria and chloroplasts are essential organelles that carry out the fundamental cellular process of respiration and photosynthesis, respectively. Organelles possess their own genomes that are physically separated from the nuclear genes, and mutations in their genome lead to serious consequences on organismal homeostasis and viability e.g. flower sterility in plants and mitochondrial myopathies in humans. However, contrary to the nucleus, methods for the genetic engineering of organellar genomes in multicellular organisms are either lacking or too laborious to be widely applied. Thus, there is a need for the development of such tools. The overall objectif of the project is to establish a new tool for the manipulation of organellar genomes in plants by exploiting the largest family of organelle RNA binding proteins in eukaryotes, the PPR family. We propose to engineer the RNA binding specificity of synthetic PPR tracts to bind specified mRNA genes in Arabidopsis organelles in order to control the expression of their cognate gene targets in vivo. We reported the in vivo functions of a synthetic PPR protein, made of consensus PPR motifs that were designed to bind a sequence near the 5’ end of a transcript in Arabidopsis chloroplasts. We used a functional complementation assay to demonstrate that this protein bound its intended RNA target with specificity in vivo and that it substituted for a natural PPR protein by stabilizing its cognate processed mRNA. Our results showed that synthetic PPRs can be engineered to functionally mimic the class of native PPR proteins that serve as physical barriers against exoribonucleases. Our work provided example for the use of synthetic PPR proteins for the control of organellar gene expression in plants.
In this 2-year project, we successfully engineered four customized PPR RNA binding proteins with desired chloroplast RNA sequence binding specificity using concatenation of a synthetic PPR motif whose RNA base recognition could be programmed using a combinatorial amino acid code (Shen C, et al 2015, Mol Plant). Before testing the in planta activity of these 4 SynPPRs, their specific RNA sequence binding was confirmed in vitro by expressing and purifying E. coli recombinant SynPPRs and conducting gel electromobility shift assays. We tested the in vivo activity of these proteins by stably expressing them in Arabidopsis plants. We confirmed the chloroplast localization for all 4 SynPPRs in plant cells by transient expression of the GFP fusion proteins in tobacco leaf cells and confocal microscopy. We showed that each protein localizes in chloroplasts where they associate with the nucleoids, a sub-compartment were posttranscriptional processes take place. Arabidopsis transgenic lines were obtained for the four transgenes and selected for phenotypic analyses but western-blot analyses demonstrated that only one of four SynPPRs was overexpressing in plants. Therefore, we tested the effect of this SynPPR overexpression on its cognate gene target in chloroplasts. We demonstrated that the SynPPR had the in vivo capacity to define a new 5’-end for its mRNA target. We confirmed by polysome analysis and in vivo 35S-Met pulse labelling that the mRNA isoform with a new 5’-end was efficiently translated in the transgenic plants. Furthermore, we performed RIP-seq analysis on transgenic plants and showed that the SynPPR protein binds preferentially to its defined rbcL RNA binding site in vivo. Our results demonstrated that synthetic PPR scaffolds hold in vivo capacities similar to those of natural PPRs by sequestrating specific RNA sequences and preventing their access to exoribonucleases. These results provide example for the use of the PPR technology for the targeted control of RNA metabolism in plant organelles. A manuscript is being finalized for publication in a scientific journal in open access mode and the NGS data (RIP-seq) were submitted to the GEO repository.
The manipulation of organellar genomes is a major challenge for the basic and applied research and there is an urgent need for the development of easy methods to achieve this. Our project has tackled this challenge in the model plant Arabidopsis by implementing an innovative approach and simple methodologies that employ designer PPRs, a recently recognized family of organellar RNA binding proteins that shows particular promise for protein engineering and the targeted control of gene expression in organelles. We can now rationally design artificial PPR repeats that bind specified RNAs in vitro and we have reported the activity and potential applications of synthetic PPRs in plants. As such, our approach offers the potential to overcome the limit of organelle genetic transformation by employing engineered nuclear encoded proteins. Such technology has the prospect of being implementable in most research labs. Finally, the originality of the project resided in the concerted approaches of state-of-the-art molecular and cellular biology, protein biochemistry and genetic techniques to deepen the possibility of genetic engineering in living organisms
Structure of a synthetic PPR protein