Targets and functions of the chloroplast ribonucleoproteins CP33A and CP33B
Final Report Abstract
The aim of this project was to investigate the functions of chloroplast RNA-binding proteins (cpRNPs) in plants, particularly their role in the post-transcriptional regulation of gene expression. Chloroplasts possess their own genome, whose expression must be precisely regulated. The stabilization and processing of RNA plays a crucial role in this process. cpRNPs are proteins that bind to RNA molecules and thereby influence their stability, maturation, or translation. They respond dynamically to environmental stimuli, such as temperature changes. Originally, the project aimed to study the function of the cpRNP CP33A in particular. However, difficulties in generating viable mutants led to a shift in focus to the related protein CP29A. For CP29A, mutants with clearly recognizable phenotypic alterations were already available. It was found that CP29A plays a central role in the expression of the rbcL gene, which encodes the large subunit of the Rubisco enzyme - a key enzyme in photosynthesis. Especially under cold stress, CP29A supports the production of Rubisco, which is essential for plant cold acclimation. These findings are based on molecular biology techniques such as eCLIP (enhanced cross-linking and immunoprecipitation) and RNA-Bind-N-Seq, which allow high-resolution analysis of RNA-protein interactions. In genetically modified tobacco plants, a significant reduction in rbcL mRNA and protein levels under cold conditions was observed, underscoring the importance of CP29A. Another important finding was that CP29A undergoes so-called phase separation under cold conditions - it forms liquid-liquid separated compartments near chloroplast nucleoids. This is the first time such a phenomenon has been described in the context of gene expression in plant organelles. The structural basis for this phase separation was analyzed in collaboration with Prof. Sattler’s lab using NMR spectroscopy, which identified the importance of a prion-like domain within CP29A. In parallel, the protein CP33B was studied and identified as an RNA-binding partner of psbA mRNA. This gene encodes the D1 protein of Photosystem II, a central component of photosynthesis that is particularly susceptible to light-induced damage and must therefore be continuously synthesized. Using methods such as RIP-Seq and RBNS, it was shown that CP33B binds almost all psbA transcripts in chloroplasts, suggesting a potentially important regulatory role. However, despite this strong binding, mutants showed no obvious defects in psbA expression or photosynthetic performance, which may be due to functional redundancies with other proteins. Due to the lack of phenotypic effects, detailed functional analysis of CP33B was not pursued further. Overall, the project made a significant contribution to understanding RNA-based regulation in chloroplasts. It established new methodological standards (first-time use of eCLIP and RBNS in plant organelles), provided molecular insights into mechanisms of cold adaptation, and opened new perspectives on the biophysical properties of RNA-binding proteins in plant organelles.
Publications
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Exploring the proteome associated with the mRNA encoding the D1 reaction center protein of Photosystem II in plant chloroplasts. The Plant Journal, 102(2), 369-382.
Watkins, Kenneth P.; Williams‐Carrier, Rosalind; Chotewutmontri, Prakitchai; Friso, Giulia; Teubner, Marlene; Belcher, Susan; Ruwe, Hannes; Schmitz‐Linneweber, Christian; van Wijk, Klaas J. & Barkan, Alice
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The Chloroplast Ribonucleoprotein CP33B Quantitatively Binds the psbA mRNA. Plants, 9(3), 367.
Teubner, Marlene; Lenzen, Benjamin; Espenberger, Lucas Bernal; Fuss, Janina; Nickelsen, Jörg; Krause, Kirsten; Ruwe, Hannes & Schmitz-Linneweber, Christian
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The Chloroplast RNA Binding Protein CP29A supportsrbcLexpression during cold acclimation. American Geophysical Union (AGU).
Lenzen Benjamin, Rösch Florian; Ruwe Hannes, Kachariya Nitin; Legen Julia, Sattler Michael & Small Ian, Schmitz-Linneweber Christian
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A prion-like domain is required for phase separation and chloroplast RNA processing during cold acclimation in Arabidopsis. The Plant Cell, 36(8), 2851-2872.
Legen, Julia; Lenzen, Benjamin; Kachariya, Nitin; Feltgen, Stephanie; Gao, Yang; Mergenthal, Simon; Weber, Willi; Klotzsch, Enrico; Zoschke, Reimo; Sattler, Michael & Schmitz-Linneweber, Christian
