Project Details
Projekt
Temperature-dependent phase separation of RNA binding proteins in chloroplasts
Subject Area
Plant Genetics and Genomics
Cell Biology
Cell Biology
Term
since 2022
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 506361669
Plants as sessile organisms cannot run when challenged by environmental stress, including temperature stress. Instead, they have developed a plethora of sophisticated physiological and molecular reactions to maintain homeostasis. The importance of phase separation for plant stress response is just emerging. We found that the chloroplast RNA binding protein CP29A is capable of liquid-liquid phase separation (LLPS) in vivo and in vitro. Phase separation is induced by low temperatures, fully reversible after return to ambient temperature and depends on the presence of CP29A’s prion-like domain (PLD). Disruption of CP29A or deletion of the PLD leads to leaf bleaching and compromised photosynthetic activity in the cold. eCLIP and RNA-bind-and-seq experiments identified multiple chloroplast RNA targets of CP29A, including the rbcL mRNA. Mutants of CP29A show a strong reduction of rbcL mRNA in the cold. NMR and biophysical analyses of recombinant CP29A confirm low temperature induced LLPS and provide insight into molecular interactions in the condensed phase at residue level detail. Moreover, NMR analysis of CP29As two individual RRM motifs identified surprising differences in their RNA binding properties. Encouraged by our findings with CP29A, we asked whether other chloroplast RBPs are also capable of LLPS. Using a combination of in silico predictions, in vivo transient expression analysis and in vitro LLPS assays, we present evidence for two proteins involved in chloroplast RNA editing and three proteins required for chloroplast translation capable of LLPS. We now propose to follow up these first descriptions of functional LLPS of chloroplast RBPs with (i) a detailed analysis of the biochemical and biophysical mechanisms underlying CP29A LLPS, and (ii) by a general characterization of the function of LLPS for the additional chloroplast RNA binding proteins residing in granules discovered here. For this goal, we bring together our complementary expertise in plant genetics and plant RNA biology (Schmitz-Linneweber lab) with expertise in the analysis of macromolecule structure, dynamics and interactions (Sattler lab). We believe that a strength of our approach are synergies from linking detailed biophysical and biochemical exploration of LLPS with molecular and in particular organismic phenotypes for plants. Furthermore, we are set up to determine the details of what allows LLPS to occur in response to a cold-shift. Finally, LLPS has rarely been analyzed in plants, despite the fact that plants as sessile organisms would greatly benefit from LLPS is a regulatory option to adjust to environmental changes. We are in a position to fill this gap for a number of chloroplast proteins.
DFG Programme
Priority Programmes
