Plant regulatory networks : RNA binding proteins as mediators of communication between DNA containing compartments by dual targeting

Abstract

The endosymbiosis-derived organelles within a plant cell, plastids and mitochondria, have to be equipped with a certain set of proteins to be fully functional. This set of proteins is encoded by different genomes: the organellar genomes and the nuclear genomes. This setup poses some interesting challenges for the regulation of gene expression and protein transport. On the one hand, the targeting signals that transport proteins to the organelles have to be highly specific and on the other hand, the communication between the DNA containing compartments to coordinate their gene expression has to be transmitted somehow, not only from the nucleus but also from the organelles back to the nucleus. In this thesis, two prediction programs are introduced. One of them can predict dual targeted proteins to both organelles (ATP, ambiguous targeting predictor) and the other one is species- specific for Physcomitrella patens (GTP_Pp; green targeting predictor – P. patens-specific). The first predictor can help to gain a more complete picture of the proteins potentially present in the organelles. With the help of that predictor, we predicted that the amount of proteins with dual targeting signals is higher than anticipated and that we to date know only a minor part of actually dual targeted proteins. The second predictor can help to answer the question on the evolutionary consistency of targeting signals within the plant kingdom and the importance of having species-specific approaches in analyzing protein targeting. We actually observed a surprisingly big difference in composition and recognition of mitochondrial and dual-targeting protein signals, which led to the conclusion, that species-specific approaches always should be considered as the optimal option for both, in silico and in vivo experiments. The second part of this thesis focuses the mechanisms of communication between nucleus and the organelles, especially the plastid possibly mediated by dual targeting. We chose several plastid RNA-/DNA-binding proteins to analyze their sub-plastidic localization and their potential additional nuclear localization. Those candidates were AtWHY1 (Arabidopsis thaliana Whirly1), four members of the AtcpRNP (chloroplast ribonucleoprotein) family and AtEF-Tu (elongation factor thermo-unstable). The analyzed members of the AtcpRNP family reflect their described multiple functions within the plastid also in a multiple localization pattern. Furthermore, we were able to show interactions of different members of the AtcpRNP family by yeast-two-hybrid interaction assays. The localization pattern of AtEF-Tu was very similar to the one observed for the AtcpRNPs, which indicated, together with a confirmed localization within the transcriptionally active chromosome, a multiple function for AtEF-Tu. The sub-plastidic localization data suggest overlapping networks of activity for the proteins by observed co-localizations. This was also shown with respect to several marker proteins for plastid functions. For AtEF-Tu and the AtcpRNPs, we also showed experimentally that a second localization in the nucleus is possible for the mature protein, which makes them interesting candidates for a possible mediation of plastid signals to the nucleus next to AtWHY1. For AtWHY1, we were able to show an effect of the DNA binding domain on the known localization pattern which seemed to reflect an aberration in transport processes through the envelope. This offers a potential regulatory mechanism that needs to be explored in detail in the future.