In mitochondria of higher plants, the majority of 59 termini of mature mRNAs are generated posttranscriptionally. To gain insight into this process, we analyzed a natural 59 end polymorphism in the species Arabidopsis thaliana. This genetic approach identified the nuclear gene At1g62670, encoding a pentatricopeptide repeat protein. The functional importance of this mitochondrial restorer of fertility-like protein, designated RNA PROCESSING FACTOR2 (RPF2), is confirmed by the analysis of a respective T-DNA knockout mutant and its functional restoration by in vivo complementation. RPF2 fulfills two functions: it is required for the generation of a distinct 59 terminus of transcripts of subunit 9 of the NADH DEHYDRO-GENASE complex (nad9) and it determines the efficiency of 59 end formation of the mRNAs for subunit 3 of the CYTOCHROME C OXIDASE (cox3), the latter also being influenced by mitochondrial DNA sequences. Accordingly, recombinant RPF2 protein directly binds to a nad9 mRNA fragment in vitro. Two-dimensional gel electrophoresis and immunodetection analyses reveal that altered 59 processing does not influence accumulation of the nad9 and cox3 polypeptides. In accessions C24, Oystese-1, and Yosemite-0, different inactive RPF2 alleles exist, demonstrating the variability of this gene in Arabidopsis. The identification of RPF2 is a major step toward the characterization of 59 mRNA processing in mitochondria of higher plants.

SUMMARYProcessing of 5¢-ends is a frequently observed step during maturation of plant mitochondrial mRNAs. Up to now, very little is known about the biochemistry of this process and the proteins involved in the removal of 5¢ leader sequences. Based on natural genetic variation we have used linkage mapping and complementation studies to identify a nuclear gene required for the efficient generation of a 5¢-end 228 nucleotides upstream of the mitochondrial nad4 gene encoding subunit 4 of the NADH dehydrogenase complex. This nuclear gene, At1g12700, that we designate RNA PROCESSING FACTOR 1 (RPF1), encodes a pentatricopeptide repeat (PPR) protein of the P-class containing canonical PPR-repeats. RPF1 belongs to a subgroup of PPR proteins, which includes the RESTORER OF FERTILITY (RF) gene products restoring cytoplasmic male sterility (CMS) in various plant species. CMS is a mitochondrially inherited trait caused by the expression of aberrant, chimeric genes, which has not been observed in the predominantly inbreeding species Arabidopsis thaliana. The here reported results are a further step towards the characterization of the plant mitochondrial RNA processing machinery and provide additional insights into the function of RF-like PPR proteins.

RNA PROCESSING FACTOR1 (RPF1) and RPF2 are pentatricopeptide repeat (PPR) proteins involved in 5# processing of different mitochondrial mRNAs in Arabidopsis (Arabidopsis thaliana). Both factors are highly similar to RESTORERS OF FERTILITY (RF), which are part of cytoplasmic male sterility/restoration systems in various plant species. These findings suggest a predominant role of RF-like PPR proteins in posttranscriptional 5# processing. To further explore the functions of this group of proteins, we examined a number of T-DNA lines carrying insertions in the corresponding PPR genes. This screening identified a nearly complete absence of mature ccmC transcripts in an At1g62930 T-DNA insertion line, a phenotype that could be restored by the introduction of the intact At1g62930 gene into the mutant. The insertion in this nuclear gene, which we now call RPF3, also leads to a severe reduction of the CcmC protein in mitochondria. The analysis of C24/rpf3-1 F2 hybrids lacking functional RPF3 genes revealed that this gene has less influence on the generation of the mature ccmC 5# transcript end derived from a distinct ccmC 5# upstream configuration found in mitochondrial DNAs from C24 and other accessions. These data show that a particular function of an RF-like protein is required only in connection with a distinct mtDNA configuration. Our new results further substantiate the fundamental role of RF-like PPR proteins in the posttranscriptional generation of plant mitochondrial 5# transcript termini.

In our analysis of 5# and 3# end formation in plant mitochondria, we compared the major transcript ends of all mitochondrial protein-coding genes between the three Arabidopsis (Arabidopsis thaliana) accessions Columbia (Col), C24, and Landsberg erecta (Ler). Differences between transcript patterns were found for seven genes. For atp6-2, no transcripts at all were detected in Ler. This and further analyses suggest that the atp6-2 gene arrangement is absent from the mitochondrial DNA of this accession. All other transcript polymorphisms are attributed to variations at the 5# termini and were consistently observed in all tissues investigated. mRNA phenotyping of reciprocal Col/Ler, Col/C24, and Ler/C24 F 1 hybrids revealed the differing transcript patterns of ccmC to be inherited maternally, suggesting these to arise from differences in the mitochondrial DNA. Biparental inheritance was observed for the polymorphic transcripts of nad4, nad9, ccmB, and rpl5, indicating these differences to be caused by nuclear-encoded trans-factors. Deviant transcript patterns were tested in further accessions and were found in at least three additional accessions. Detailed examination of the nad4 and the nad9 transcripts demonstrates that the respective polymorphisms affect the major mRNAs of these genes. This study shows that natural genetic variation in Arabidopsis can also affect mitochondrial mRNA end processing. These variations can now be used to identify the nuclear genes responsible, as well as the mitochondrial cis-elements required, for 5# end generation of mitochondrial transcripts.

SUMMARYThe 5′ ends of many mitochondrial transcripts are generated post-transcriptionally. Recently, we identified three RNA PROCESSING FACTORs required for 5′ end maturation of different mitochondrial mRNAs in Arabidopsis thaliana. All of these factors are pentatricopeptide repeat proteins (PPRPs), highly similar to RESTORERs OF FERTILTY (RF), that rescue male fertility in cytoplasmic male-sterile lines from different species. Therefore, we suggested a general role of these RF-like PPRPs in mitochondrial 5′ processing. We now identified RNA PROCESSING FACTOR 5, a PPRP not classified as an RF-like protein, required for the efficient 5′ maturation of the nad6 and atp9 mRNAs as well as 26S rRNA. The precursor molecules of these RNAs share conserved sequence elements, approximately ranging from positions À50 to +9 relative to mature 5′ mRNA termini, suggesting these sequences to be at least part of the cis elements required for processing. The knockout of RPF5 has only a moderate influence on 5′ processing of atp9 mRNA, whereas the generation of the mature nad6 mRNA and 26S rRNA is almost completely abolished in the mutant. The latter leads to a 50% decrease of total 26S rRNA species, resulting in an imbalance between the large rRNA and 18S rRNA. Despite these severe changes in RNA levels and in the proportion between the 26S and 18S rRNAs, mitochondrial protein levels appear to be unaltered in the mutant, whereas seed germination capacity is markedly reduced.

Natural genetic variation affects development, physiology, biochemical properties as well as mitochondrial transcripts of the model species Arabidopsis thaliana (Arabidopsis). In a previous study, we identified mitochondrial transcript end polymorphisms in Arabidopsis accessions Columbia, C24 and Landsberg erecta. The polymorphic transcript species could either be assigned to differences in the mitochondrial DNA or to natural genetic variation in the nucleus. To analyze the distribution and to identify additional 5' end polymorphisms we now analyzed 19 mitochondrial transcription units in 26 different accessions. We found additional 5' end polymorphisms indicating that such transcript length differences are a widespread phenomenon in Arabidopsis. The new polymorphisms affect cox1, cox2, nad2 as well nad3-rps12 transcript species. While the cox2 polymorphism can be attributed to a recombination event in the mitochondrial DNA, the nad2 transcript polymorphism is linked to differences in the nuclear DNA. A complex pattern is found for nad3-rps12 mRNA whose 5' ends differ between several accessions. These new polymorphisms provide an important basis for a more detailed characterization of mitochondrial 5' end processing.

SUMMARYPlant mitochondrial transcripts undergo maturation processes at both termini. Although frequently observed, the post-transcriptional formation of mature 5 0 ends is still poorly understood. We now analyzed the processing of transcripts derived from the mitochondrial ccmC gene, coding for a component of the cytochrome c maturation system. In Arabidopsis thaliana (Arabidopsis) there are two mitochondrial ccmC configurations, discriminated by a 66-bp sequence segment located approximately 500 bp upstream of the ccmC gene. In Arabidopsis accessions these divergent mitochondrial genotypes correlate with the generation of two different 5 0 termini that map to positions around À484 in accession Columbia (Col ccmC genotype) or À390 in accession C24 relative to the translation start codon (C24 ccmC genotype). Previously we identified RNA PROCESSING FACTOR 3 (RPF3), a pentatricopeptide repeat (PPR) protein required for the maturation of ccmC mRNAs with À484 5 0 ends transcribed from the Col ccmC genotype. Now we identified several accessions defective in maturation of ccmC transcripts. Taking advantage of this natural genetic variation we identified RNA PROCESSING FACTOR 6 (RPF6), a PPR protein necessary for the generation of ccmC mRNAs with À390 5 0 ends transcribed from the C24 ccmC genotype. Both Col-type and C24-type accessions encode RPF3 and RPF6 so that they can process ccmC transcripts derived from the two different mitochondrial genotypes. These factors and their cognate RNA recognition sites in the different ccmC genotypes are an intriguing example for the evolution of two co-adapted cyto-nuclear systems required for the same process i.e. 5 0 maturation of ccmC transcripts.

In plant mitochondria, the 5' ends of many transcripts are generated post-transcriptionally. We show that the pentatricopeptide repeat (PPR) protein RNA PROCESSING FACTOR 4 (RPF4) supports the generation of extra 5' ends of ccmB transcripts in Landsberg erecta (Ler) and a number of other Arabidopsis thaliana ecotypes. RPF4 was identified in Ler applying a forward genetic approach supported by complementation studies of ecotype Columbia (Col), which generates the Ler-type extra ccmB 5' termini only after the introduction of the RPF4 allele from Ler. Studies with chimeric RPF4 proteins composed of various parts of the RPF4 proteins from Ler and Col identified differences in the N-terminal and central PPR motifs that explain ecotype-specific variations in ccmB processing. These results fit well with binding site predictions in ccmB transcripts based on the known determinants of nucleotide base recognition by PPR motifs.