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A Short 101 of Everything

Green as a ... Chloroplast!
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Our group is fascinated by the arguably most important organelle on the planet: the chloroplast. Host to the photosynthetic apparatus, chloroplasts provide plant cells not only with reduced carbon, but also with a wealth of other metabolites (nucleotides, amino acids, fatty acids, vitamins, etc., etc.).
Strikingly, this organelle, although a defining feature of the eukaryotic plant cell, is not a eukaryotic invention. Instead, chloroplasts number cyanobacteria among their closest ancestry. Thus, chloroplasts are undercover-prokaryotes that have given up their independence and became endosymbionts of the eukaryotic host cell.

Chloroplast Gene Expression
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Since the initial uptake about 1.5 billion years ago, chloroplasts have lost most of their genetic information. A large number of their genes have been transferred to the host genome, but a small number is retained; in seed plants typically 110 genes, deposited on a chromosome of about 150 kb.
 
This genome has to be expressed, and one of the most interesting features of plastid gene expression is the complexity of posttranscriptional events. Plastid RNAs – most of them polycistronic - are cleaved internally, trimmed at the ends, spliced and edited. These processes are all mediated by nuclear-encoded factors that are imported posttranslationally into chloroplasts and obviously, many of them have to be RNA-binding proteins.

Chloroplast RNA Binding Proteins
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There is a broad variety of putative RNA binding proteins coded in plant nuclear genomes that are predicted to be targeted to the chloroplast. Their functional assignment is usually based on their known RNA binding motifs and not on experimental evidence. In the Arabidopsis and rice genomes many plastid-targeted proteins with RRM-, Zn finger-, KH-, S1, RNAseIII-, etc-domains have been described.

Some of these proteins will be specific to one or very few messages while others target all or a larger subset of plastid RNAs. This allows the functional and spatial partition of the cellular transcriptome into distinct subsets. Ad extremo, such a subset can consist of only a single message as exemplified by specific splice-factors bound to a single or very few RNAs (e.g. the splice factor CRS1).

On the other hand, much larger subsets are defined by general RNA binding proteins, the ribosome or the basal splice machinery. Both, specific and general interactions are needed to adjust the plant transcriptome to tissue- and time-specific needs. An example for an RNA binding protein family with members that are believed to be highly specific for their respective targets is the PPR protein family.

PPR Proteins: a Large Plant-Specific RNA Binding Protein Family
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PPR (pentatricopeptide repeat) proteins have a broad phylogenetic distribution, but the family is highly expanded specifically in land plants. Roughly 450 PPR proteins are predicted in Arabidopsis and rice and about 80% of all PPRs are expected to be targeted to mitochondria and chloroplasts.
The PPR motif is a degenerate 35 amino acid repeat that likely forms a helical hairpin repeat protein domain. Current models suggest that consecutive PPR domains are stacked and form a surface with a characteristic charge distribution that allows interaction with target RNAs. A variety of genetic data suggest that PPRs are important for the maturation of specific organellar transcripts. However, only very few PPR proteins have been tested for RNA interaction partners, most of them in vitro.

cpRNP Proteins: Light-to-RNA Signal transducers
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The cpRNPs are highly abundant, general RNA-binding proteins of the RRM-type and are related to eukaryotic hnRNP proteins. In previous in vitro studies, cpRNPs have been shown to interact with ribosome-free mRNAs, to increase the half life of the mRNA pool [1], and to be required for RNA editing [2]. Notably, cpRNPs are regulated via posttranslational modifications in a light dependent fashion [3].
By analyzing Arabidopsis mutants, we found that cp31A, one of eight cpRNPs in Arabidopsis thaliana, is required for the accumulation of a single chloroplast mRNA. In addition, cp31A and its paralogue cp31B contribute in a redundant or additive fashion to the processing of several RNA editing sites. Our current research focuses on the molecular functions of the cpRNPs in the maturation and stabilization of chloroplast mRNAs. Furthermore, we are investigating if cpRNPs modulate the chloroplast transcriptome in response to environmental cues like light.

  • [1] Nakamura T, Ohta M, Sugiura M, Sugita M (2001): Chloroplast ribonucleoproteins function as a stabilizing factor of ribosome-free mRNAs in the stroma. J Biol Chem. 276(1):147-52.
  • [2] Hirose T, Sugiura M (2001): Involvement of a site-specific trans-acting factor and a common RNA-binding protein in the editing of chloroplast mRNAs: development of a chloroplast in vitro RNA editing system. Embo J. 20(5): 1144-52.
  • [3] Bai-Chen W, Hong-Xia W, Jian-Xun F, Da-Zhe M, Li-Jia Q, Yu-Xian Z (2006): Post-translational modifications, but not transcriptional regulation, of major chloroplast RNA-binding proteins are related to Arabidopsis seedling development. Proteomics 6(8): 2555-2563.

MatK: a polyvalent chloroplast-encoded intron maturase
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Organellar group II introns have been considered to be the likely ancestors of nuclear spliceosomal introns. In bacteria, maturases are encoded by group II introns, and are required for splicing of their respective home introns. In chloroplasts and mitochondria of photosynthetic land plants, most group II introns have lost maturase reading frames. We found that the essential chloroplast maturase MatK interacts not only with its home intron within trnK-UUU, but also with six additional introns, all belonging to intron subclass IIA.
Analyses of binding sites of MatK in two selected target introns demonstrate deviations from bacterial maturase-intron interactions in chloroplasts. These results demonstrate that organellar maturases are capable of associating with multiple intron ligands in a novel fashion, making them an attractive model for an early trans-acting nuclear splicing activity. Current research is directed towards understanding the interaction of MatK with its different targets. Furthermore, we would like to understand why matK has not been transferred to the nucleus and remains a chloroplast reading frame. We speculate that the development of autoregulatory circuits of matK expression immobilize the matK gene inside the chloroplast compartment.