Functions of PPR proteins

Translation remains the most elusive step of gene expression in plant organelles. The specificities of the translation apparatus in plant mitochondria are investigated by a combination of biochemical and structural approaches, i.e. by single particle cryo-electron microscopy. Results show that Arabidopsis mitoribosomes are characterized by numerous additional protein subunits, in particular PPR proteins, whose exact functions are investigated by reverse genetic methods.

Beyond PRORP proteins that hold RNase P activity, we study other NYN domain nucleases that represent the diversity of these enzymes in plants. One of them, YacP participates in the maturation of mRNAs in chloroplasts. Another enzyme, MNU2 interacts with the PRORP1 protein located in the mitochondria. The study of this NYN domain enzyme complex will help to understand its role in mitochondrial gene expression.

The integration of PRORP functions within the overall framework of the plant cell is determined through the characterization of PRORP interactomes with proteins and RNA. In order to understand the diversity and evolution of PRORP proteins, these enzymes are characterized in the green alga Chlamydomonas, the malaria parasite Plasmodium and the nematode Romanomermis. The detailed understanding of the diversity of PRORP proteins and comparisons with ribonucleoprotein RNase P are expected to give clues to understand the transition from the prebiotic RNA world to today’s world dominated by proteins.

Specificities of PPR proteins mode of action are exemplarily investigated for PRORP proteins. This project is based on biophysical analyses of these enzymes, in particular, through crystallographic and solution structure analyses of PRORP proteins alone and in complex with pre-tRNA substrates. The dynamics and kinetic parameters of this complex are also analysed with an array of biochemical and biophysical approaches.

Many plant viruses are characterized by the occurrence of essential tRNA-like structures (TLS) at the 3’ end of their genomic RNA. Because virus replication takes place in the cytosol, the targeting signal of PRORP enzymes is manipulated to induce PRORP accumulation in the cytosol and the cleavage of viral TLS, leading to an arrest of virus replication. After an anlaysis with Arabidopsis and the TYMV, this biotechnological application will be evaluated with plants of economic relevance and an array of plant viruses.