Rapid ribonucleoprotein-dependent recruitment of mRNA to ribosomes in the high light acclimation of Arabidopsis thaliana

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The proposed research aims at understanding the extremely rapid effect of chloroplast metabolic state on cytosolic translation mediated by control of ribonucleoproteins. The project takes advantage of the precise experimental design of low light-to-high light- (L-H)-transfer and exploits the very fast conditional reorganization of the translatome. It is based on three hypotheses namely that (i) chloroplasts control the translational activity of ribosomes in their cellular vicinity, (ii) that the massive reorganization of ribosome-loaded mRNAs upon L-H-transfer depends on features of the UTR and RNA-binding proteins (RBPs) and (iii) that posttranslational modifications of RBPs determine the recruitment of specific mRNA to the ribosomes. The three hypotheses will be addressed in seven work packages. The stress associated Zinc-finger protein SAP3 will be used as an example to test the specificity and function of three sequence motifs found to be highly overrepresented in the 5-UTR among the 272 transcripts preferentially associated with the polysomes 10 min after L-H-transfer. Using RNAseq on polysomal RNAs we will refine the data set of rapid ribosome-recruited mRNAs and the reversibility of this regulatory process. The significance of the three motifs and the 5-UTR and 3-UTR in H-L-transfer will be scrutinized by using transfected protoplasts and stably transformed sap2/sap3-double mutants of Arabidopsis thaliana. RBPs will be identified that bind to the motifs and the UTRs and dynamic differences between the polysomal proteomes of low light and L-H-samples will be identified. The molecular and physiological role of identified RBPs will be investigated in vitro und in vivo. In order to complete the understanding of the involved pathways and signals, upstream coupling of the translational L-H-regulation to the MAP kinase pathway will be addressed using defined genetic mutants, e.g. mpk6. Regulation of downstream target genes like the sigma factor 5 and small HSPs will be assessed by physiological analysis of knock out plants. This project is integrated into the framework of SPP1935 according to the specific aims and work plan defined in the call, i.e. clarification of regulation and dynamics of RNPs, establishment and application of technologies for specific RNP isolation and identification, and expansion of the specific regulatory network including upstream and downstream elements in context of the fundamental process of light acclimation in higher plants.