OTP70 is a pentatricopeptide repeat protein of the E subgroup involved in splicing of the plastid transcript rpoC1.

Over 20 proteins of the pentatricopeptide repeat (PPR) family have been demonstrated to be involved in RNA editing in plant mitochondria and chloroplasts. All of these editing factors contain a so-called 'E' domain that has been shown to be essential for editing to occur. The presumption has been that this domain recruits the (unknown) editing enzyme to the RNA. In this report, we show that not all putative E-class PPR proteins are directly involved in RNA editing. Disruption of the OTP70 gene leads to a strong defect in splicing of the plastid transcript rpoC1, leading to a virescent phenotype. The mutant has a chloroplast transcript pattern characteristic of a reduction in plastid-encoded RNA polymerase activity. The E domain of OTP70 is not required for splicing, and can be deleted or replaced by the E domain from the known editing factor CRR4 without loss of rpoC1 splicing. Furthermore, the E domain of OTP70 is incapable of inducing RNA editing when fused to the RNA binding domain of CRR4. We conclude that the truncated E domain of OTP70 is no longer functional in RNA editing, and that the protein has acquired a new function in promoting RNA splicing.

Arabidopsis CSP41 proteins form multimeric complexes that bind and stabilize distinct plastid transcripts.

The spinach CSP41 protein has been shown to bind and cleave chloroplast RNA in vitro. Arabidopsis thaliana, like other photosynthetic eukaryotes, encodes two copies of this protein. Several functions have been described for CSP41 proteins in Arabidopsis, including roles in chloroplast rRNA metabolism and transcription. CSP41a and CSP41b interact physically, but it is not clear whether they have distinct functions. It is shown here that CSP41b, but not CSP41a, is an essential and major component of a specific subset of RNA-binding complexes that form in the dark and disassemble in the light. RNA immunoprecipitation and hybridization to gene chips (RIP-chip) experiments indicated that CSP41 complexes can contain chloroplast mRNAs coding for photosynthetic proteins and rRNAs (16S and 23S), but no tRNAs or mRNAs for ribosomal proteins. Leaves of plants lacking CSP41b showed decreased steady-state levels of CSP41 target RNAs, as well as decreased plastid transcription and translation rates. Representative target RNAs were less stable when incubated with broken chloroplasts devoid of CSP41 complexes, indicating that CSP41 proteins can stabilize target RNAs. Therefore, it is proposed that (i) CSP41 complexes may serve to stabilize non-translated target mRNAs and precursor rRNAs during the night when the translational machinery is less active in a manner responsive to the redox state of the chloroplast, and (ii) that the defects in translation and transcription in CSP41 protein-less mutants are secondary effects of the decreased transcript stability.

Phage-type RNA polymerase RPOTmp performs gene-specific transcription in mitochondria of Arabidopsis thaliana.

Transcription of mitochondrial genes in animals, fungi, and plants relies on the activity of T3/T7 phage-type RNA polymerases. Two such enzymes, RPOTm and RPOTmp, are present in the mitochondria of eudicotyledonous plants; RPOTmp is additionally found in plastids. We have characterized the transcriptional role of the dual-targeted RNA polymerase in mitochondria of Arabidopsis thaliana. Examination of mitochondrial transcripts in rpoTmp mutants revealed major differences in transcript abundances between wild-type and rpoTmp plants. Decreased levels of specific transcripts were correlated with reduced abundances of the respiratory chain complexes I and IV. Altered transcript levels in rpoTmp were found to result from gene-specific transcriptional changes, establishing that RPOTmp functions in distinct transcriptional processes within mitochondria. Decreased transcription of specific genes in rpoTmp was not associated with changes in promoter utilization; therefore, RPOTmp function is not promoter specific but gene specific. This implies that additional gene-specific elements direct the transcription of a subset of mitochondrial genes by RPOTmp.

The pentatricopeptide repeat gene OTP51 with two LAGLIDADG motifs is required for the cis-splicing of plastid ycf3 intron 2 in Arabidopsis thaliana.

Summary The Arabidopsis thaliana chloroplast contains 20 group-II introns in its genome, and seven known splicing factors are required for the splicing of overlapping subsets of 19 of them. We describe an additional protein (OTP51) that specifically promotes the splicing of the only group-II intron for which no splicing factor has been described previously. This protein is a pentatricopeptide repeat (PPR) protein containing two LAGLIDADG motifs found in group-I intron maturases in other organisms. Amino acids thought to be important for the homing endonuclease activity of other LAGLIDADG proteins are missing in this protein, but the amino acids described to be important for maturase activity are conserved. OTP51 is absolutely required for the splicing of ycf3 intron 2, and also influences the splicing of several other group-IIa introns. Loss of OTP51 has far-reaching consequences for photosystem-I and photosystem-II assembly, and for the photosynthetic fluorescence characteristics of mutant plants.

Nuclearly encoded splicing factors implicated in RNA splicing in higher plant organelles.

Plant organelles arose from two independent endosymbiosis events. Throughout evolutionary history, tight control of chloroplasts and mitochondria has been gained by the nucleus, which regulates most steps of organelle genome expression and metabolism. In particular, RNA maturation, including RNA splicing, is highly dependent on nuclearly encoded splicing factors. Most introns in organelles are group II introns, whose catalytic mechanism closely resembles that of the nuclear spliceosome. Plant group II introns have lost the ability to self-splice in vivo and require nuclearly encoded proteins as cofactors. Since the first splicing factor was identified in chloroplasts more than 10 years ago, many other proteins have been shown to be involved in splicing of one or more introns in chloroplasts or mitochondria. These new proteins belong to a variety of different families of RNA binding proteins and provide new insights into ribonucleo-protein complexes and RNA splicing machineries in organelles. In this review, we describe how splicing factors, encoded by the nucleus and targeted to the organelles, take part in post-transcriptional steps in higher plant organelle gene expression. We go on to discuss the potential for these factors to regulate organelle gene expression.

The pentatricopeptide repeat gene OTP43 is required for trans-splicing of the mitochondrial nad1 Intron 1 in Arabidopsis thaliana.

The mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) is a large protein complex formed from both nuclearly and mitochondrially encoded subunits. Subunit ND1 is encoded by a mitochondrial gene comprising five exons, and the mature transcript requires four RNA splicing events, two of which involve trans-splicing independently transcribed RNAs. We have identified a nuclear gene (OTP43) absolutely required for trans-splicing of intron 1 (and only intron 1) of Arabidopsis thaliana nad1 transcripts. This gene encodes a previously uncharacterized pentatricopeptide repeat protein. Mutant Arabidopsis plants with a disrupted OTP43 gene do not present detectable mitochondrial Complex I activity and show severe defects in seed development, germination, and to a lesser extent in plant growth. The alternative respiratory pathway involving alternative oxidase is significantly induced in the mutant.

Two members of the Arabidopsis CLC (chloride channel) family, AtCLCe and AtCLCf, are associated with thylakoid and Golgi membranes, respectively.

Though numerous pieces of evidence point to major physiological roles for anion channels in plants, progress in the understanding of their biological functions is limited by the small number of genes identified so far. Seven chloride channel (CLC) members could be identified in the Arabidopsis genome, amongst which AtCLCe and AtCLCf are both more closely related to bacterial CLCs than the other plant CLCs. It is shown here that AtCLCe is targeted to the thylakoid membranes in chloroplasts and, in agreement with this subcellular localization, that the clce mutants display a phenotype related to photosynthesis activity. The AtCLCf protein is localized in Golgi membranes and functionally complements the yeast gef1 mutant disrupted in the single CLC gene encoding a Golgi-associated protein.

Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications.

Microarray transcript profiling and RNA interference are two new technologies crucial for large-scale gene function studies in multicellular eukaryotes. Both rely on sequence-specific hybridization between complementary nucleic acid strands, inciting us to create a collection of gene-specific sequence tags (GSTs) representing at least 21,500 Arabidopsis genes and which are compatible with both approaches. The GSTs were carefully selected to ensure that each of them shared no significant similarity with any other region in the Arabidopsis genome. They were synthesized by PCR amplification from genomic DNA. Spotted microarrays fabricated from the GSTs show good dynamic range, specificity, and sensitivity in transcript profiling experiments. The GSTs have also been transferred to bacterial plasmid vectors via recombinational cloning protocols. These cloned GSTs constitute the ideal starting point for a variety of functional approaches, including reverse genetics. We have subcloned GSTs on a large scale into vectors designed for gene silencing in plant cells. We show that in planta expression of GST hairpin RNA results in the expected phenotypes in silenced Arabidopsis lines. These versatile GST resources provide novel and powerful tools for functional genomics.