Analysis of the activation of chloroplast translation and RNA stabilization by PPR proteins and PPR protein derived sRNAs
Final Report Abstract
Chloroplasts and mitochondria of land plants contain their own genetic information that is read and turned into proteins by a machinery consisting of hundreds of nuclear-encoded proteins. A large number of these proteins is required for the maturation of organellar RNAs, a process essential for setting up the photosynthetic and respiratory apparatus. More than 400 proteins from the pentatricopeptide repeat (PPR) protein family are essential players for various steps in organellar RNA processing, i.e. splicing, RNA editing, RNA stabilization and translation. PPR proteins bind tightly to specific short (15-25 nt) sequence stretches and prevent exonucleases from progressively degrading their target RNA. Eventually, PPR proteins leave behind a footprint of RNA representing their binding site. Mutants of PPR protein genes display massive defects in photosynthetic or respiratory ability, usually resulting in severe phenotypes, often in embryonic death. Within this project, we have systematically characterized PPR protein footprints by sequencing small RNAs from Arabidopsis. This work demonstrated that PPR proteins bind short footprint RNAs in vivo and are in fact titrated away from their native targets if footprints are artificially overexpressed. This prompted us to propose a novel mechanism of gene regulation in chloroplasts based on PPR protein titration. Furthermore, footprint features, like the sharpness of footprint ends, allowed us to come to general conclusions about the RNA degradation machinery in chloroplasts versus mitochondria, namely that mitochondria have a distinct way to process the 5’-termine of their RNAs independent of exonucleases. From the start of the project, we had been in particular interested in the role of PPR proteins as RNA stabilizers and translation factors, since these processes are believed to have regulatory impact on the production of the phostosynthetic and respiratory proteins. A big gap in our knowledge is how PPR proteins mediate translational activation and what the molecular details are of RNA stabilization. For this, we have generated plant lines with reporter gene constructs inserted within the chloroplast genome to test, how the position of the target site and RNA structure context of a particular well-studied PPR protein (PPR10) affects RNA stability and translation. We found that various footprint sequences of PPR proteins can be used to efficiently drive transgene expression and that the distance of the footprint to the start codon plays a role for translation, although it came as a surprise that even when spaced more than 100 nt, PPRs still support translation, arguing for yet unknown ways of translational activation. Our findings have implications for biotechnological applications, since we can use PPR footprints to manipulate expression of transgenes in the chloroplasts. Indeed, in collaboration with industrial partners, we have started expressing vaccines in the chloroplast based on PPR footprints. Time will tell, whether this approach will win recognition in chloroplast biotechnology.
Publications
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A Nucleus-Encoded Chloroplast Phosphoprotein Governs Expression of the Photosystem I Subunit PsaC in Chlamydomonas reinhardtii. Plant Cell. 2016 April 25, 28(5):1182-99
Douchi D, Qu Y, Longoni P, Legendre-Lefebvre L, Johnson X, Schmitz- Linneweber C, Goldschmidt-Clermont M
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Systematic analysis of plant mitochondrial and chloroplast small RNAs suggests organelle-specific mRNA stabilization mechanisms. Nucleic Acids Res. 2016 May 27, pp. 1-12
Ruwe H, Wang G, Gusewski S, Schmitz-Linneweber C
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CRP1 Protein: (dis)similarities between Arabidopsis thaliana and Zea mays. Front Plant Sci. 2017 Feb 15;8:163
Ferrari R, Tadini L, Moratti F, Lehniger MK, Costa A, Rossi F, Colombo M, Masiero S, Schmitz-Linneweber C, Pesaresi P
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Identification of clustered organellar short (cos) RNAs and of a conserved family of organellar RNA-binding proteins, the heptatricopeptide repeat proteins, in the malaria parasite. Nucleic Acids Res. 2018 Aug 8
Hillebrand A, Matz JM, Almendinger M, Müller K, Matuschewski K, Schmitz- Linneweber C
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Stabilization and translation of synthetic operon-derived mRNAs in chloroplasts by sequences representing PPR protein-binding sites. Plant J. 2018 Apr;94(1):8-21
Legen J, Ruf S, Kroop X, Wang G, Barkan A, Bock R, Schmitz-Linneweber C
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Unexpected functional versatility of the pentatricopeptide repeat proteins PGR3, PPR5 and PPR10. Nucleic Acids Res. 2018 Aug 16
Rojas M, Ruwe H, Miranda RG, Zoschke R, Hase N, Schmitz-Linneweber C, Barkan A
