Functional Characterization of Selected Chloroplast RNA-Binding Proteins from Arabidopsis thaliana

Abstract

Plastids are indispensable, plant-specific organelles of prokaryotic origin. They serve a plethora of biological functions crucial for plant metabolism. Plastids contain their own genomes, which are remnants of their cyanobacterial ancestor. However, mechanisms regulating expression of genes encoded in plastid DNA remain poorly understood. It is hypothesized that RNA processing plays a predominant role in this regulation but exact proteins involved in this process as well as their mode of action are not fully understood. Using a combination of phylogenetic, genetic, biochemical and cell biology approaches, I have expanded the knowledge about two groups of RNA-associated proteins, which act in Arabidopsis thaliana plastids. First, I focused on RNase H1, a conserved enzyme responsible for digestion of the RNA strand of RNA:DNA hybrids. Through the phylogenetic analysis of this protein I have shown that the common ancestor of two largest groups of land plants, monocots and dicots, contained only one RNase H1. Subsequent gene duplication occurred independently in monocots and dicots and resulted in the presence of at least two RNase H1 paralogs in most angiosperm species. Additionally, I have shown that the Arabidopsis thaliana genome contains three RNase H1 genes encoding four RNase H1 proteins which display the canonical RNase H1 activity. Furthermore, I have demonstrated that these proteins localize to the nucleus, mitochondria and chloroplasts and the presence of at least one x organellar (mitochondrial or plastid) RNase H1 is required for proper embryo development. I have also shown that plants deficient in plastid RNase H1 accumulate higher levels of plastid DNA and display elevated sensitivity to replicative stress compared to wild type plants. Altogether, these results suggest that the canonical RNase H1 activity is crucial for proper nucleic acid metabolism in plastids and for plant embryonic development. Subsequently, I have performed characterization of another family of proteins that has been previously implicated in plastid RNA processing, the Defective in Chloroplasts and Leaves (DeCL) protein family. Through phylogenetic analysis I have shown that DeCL proteins are present mostly in photosynthetic organisms and are probably of cyanobacterial origin. These proteins group into five sub-families, which share a well-defined domain of unknown function, the DeCL domain. Two of these sub-families contain subunits of plant-specific DNA-dependent RNA polymerases involved in Transcriptional Gene Silencing (RNA Pol IV and Pol V), one contains nuclear-localized rRNA binding proteins (DOMINO1) and two remaining sub-families contain organellar proteins, DeCL1 and DeCL2. I have demonstrated that in Arabidopsis thaliana both DeCL1 and DeCL2 associate with chloroplasts membranes and the presence of at least one of those proteins is required for plant viability under normal growth conditions. DeCL1 interacts with a subset of plastid-encoded mRNAs, mostly binding to their 5’ or 3’ ends. rRNAs are also bound by DeCL1 and their processing is affected in the decl1 mutant, which is consistent with previous data implicating DeCL proteins in rRNA processing. Altogether, these results provide evidence of direct or indirect DeCL-RNA interaction and suggest at least partial redundancy between DeCL1 and DeCL2. This identifies the establishment of the molecular mechanism of DeCL function as an important goal for future research. xi Altogether, this work provides important, novel insights into the processes involved in plastid RNA metabolism. It answers several important questions about two plastid RNA-associated proteins and opens new avenues for studying regulation of plastid-encoded genes at the RNA level.