PPRdesign · Engineering synthetic pentatricopeptide repeat proteins for the site-specific genetic manipulation of plant organelles
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2018-06-01 → 2020-05-31
- EU contribution
- €185,076
- Participants
- 1
- Scheme
- MSCA-IF-EF-ST
Lines connect the coordinator with its partners.
Results in brief
Engineering synthetic pentatricopeptide repeat proteins for the site-specific genetic manipulation of plant organelles
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.
Data: CORDIS, © European Union
Project objective
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. To address this issue, I will exploit nature’s design and engineer the largest protein family of organelle gene regulators found in eukaryotes, the pentatricopeptide repeat (PPR) family. PPR proteins are nuclear encoded RNA-binding proteins that function in the posttranscriptional regulation of organellar gene expression. The PPR RNA-binding domain is composed of multiple repeats of 35 amino acid long stretches. Each repeat recognizes a single base in the target RNA sequence whose identity is defined by a code involving 2 amino acids in each repeat and we can now design artificial PPR repeat proteins with programmable RNA binding specificity. The PPRdesign project will establish new methods for the site-specific control of organellar genes in the Arabidopsis model plant by targeting customized synthetic PPR repeats to specific RNA sites in chloroplasts and mitochondria.
Original text from CORDIS.
Participants
- CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS · ParisCoordinatorFrance
Links
- View on CORDIS
- DOI: 10.3030/794377
- http://www.ibmp.cnrs.fr/equipes/adaptation-genetique-du-chloroplaste/
Data: CORDIS, © European Union