Global key players of chloroplast gene expression
Plant Genetics and Genomics
Final Report Abstract
In our proposal we mainly addressed questions related to the function and the targets of RNA binding proteins as global players of the complex plastid RNA metabolism, such as 1. PAC, RNE (RNase E) and its newly identified interaction partner 2. RHON1. Furthermore, we also analysed the function of the novel factor 3. HCF145 in stabilization of plastid RNAs. We successfully studied the function of all three factors and fulfilled much more than the initial expectations as outlined in the final report. 1. The role of PAC in specific RNA binding and in accumulation of plastid ribosomes was identified. 2. In all respects, such as plastid RNA precursor accumulation, protein pattern, increased number and decreased size of chloroplasts and defective chloroplast development, the phenotype of rhonl knockout mutants resembles that of rne lines. Our data strongly suggest that RHON1 supports RNE functions presumably by conferring sequence specificity and guiding plastid RNA to RNase E and presumably additional nucleases. RNE and RHON1 form a high molecular weight complex with degradosome-like functions. We anticipate that future research on this complex, its compartmentalization and association partners will shed new light on chloroplast gene expression, division and development. The proposed additional function of RHON 1 in termination of transcription was verified. 3. The function of the novel factor HCF145 in binding and stabilization of the psaA-psaB-rps14, psbN and ycf2 mRNAs was discovered. Based on our phylogenetic and molecular analysis, we suggest that HCF145 is composed of modules of cyanobacterial origin and evolved to regulate psaA-psaB-rps14 mRNA accumulation to adjust PSI levels in an environmental and development-dependent manner. The target of HCF145 is a stretch of precisely 5 nucleotides in the psaA 5’ UTR. Binding to the 5’ UTR protects the RNA from proteolytic attack, as revealed by in vivo and in vitro nuclease protection assays as well as structural analysis. Furthermore, the newly defined, repeated RNA binding motif, designated TMR, was found not only in HCF145 but also in a variety of diverse proteins in Physcomitrella, green and red algae, as well as in cyanobacteria. In vitro binding assays with purified proteins revealed that all representative TMR proteins chosen from of all four lineages represent highly affine RNA binding proteins.
