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.

Key message RNA PROCESSING FACTORs 1 AND 8 (RPF1 and RPF8), both restorer of fertility like pentatricopeptide repeat proteins, are required for processing of dicistronic nad4L-atp4 and nad3-rps12 transcripts in Arabidopsis mitochondria. Abstract In mitochondria of Arabidopsis thaliana (Arabidopsis), the 5′ termini of many RNAs are generated on the post-transcriptional level. This process is still poorly understood in terms of both the underlying mechanism as well as proteins required. Our studies now link the generation of polymorphic 5′ extremities of the dicistronic nad3-rps12 and nad4L-atp4 transcripts to the function of the P-type pentatricopeptide repeat proteins RNA PROCESSING FACTORs 8 (RPF8) and 1 (RPF1). RPF8 is required to generate the nad3-rps12 -141 5′ end in ecotype Van-0 whereas the RPF8 allele in Col has no function in the generation of any 5′ terminus of this transcript. This observation strongly suggests the involvement of an additional factor in the generation of the -229 5′ end of nad3-rps12 transcripts in Col. RPF1, previously found to be necessary for the generation of the -228 5′ end of the major 1538 nucleotide-long nad4 mRNAs, is also important for the formation of nad4L-atp4 transcripts with a 5′ end at position -318 in Col. Many Arabidopsis ecotypes contain inactive RPF1 alleles resulting in the accumulation of various low abundant nad4L-atp4 RNAs which might represent precursor and/or degradation products. Some of these ecotypes accumulate major, but slightly smaller RNA species. The introduction of RPF1 into these lines not only establishes the formation of the major nad4L-atp4 dicistronic mRNA with the -318 5′ terminus, the presence of this gene also suppresses the accumulation of most alternative nad4L-atp4 RNAs. Beside RPF1, several other factors contribute to nad4L-atp4 transcript formation.

The Arabidopsis thaliana genome harbors more than 450 nuclear genes encoding pentatricopeptide repeat (PPR) proteins that operate in the RNA metabolism of mitochondria and/or plastids. To date, the molecular function of many PPR proteins is still unknown. Here we analyzed the nucleus-encoded gene At4g19440 coding for a P-type PPR protein. Knockout of this gene interferes with normal embryo development and seed maturation. Two experimental approaches were applied to overcome lethality and to investigate the outcome of At4g19440 knockout in adult plants. These studies revealed changes in the abundance of several mitochondria-encoded transcripts. In particular, steady-state levels of dicistronic rpl5-cob RNAs were markedly reduced, whereas levels of mature ccmC and rpl2-mttB transcripts were clearly increased. Predictions according to the one repeat to one nucleotide code for PPR proteins indicate binding of the At4g19440 protein to a previously detected small RNA at the 3 0 termini of the dicistronic rpl5-cob transcripts. This potential interaction indicates a function of this protein in 3 0 end formation and stabilization of these RNA species, whereas the increase in the levels of the ccmC mRNA along with other mitochondria-encoded RNAs seems to be a secondary effect of At4g19440 knockout. Since the inactivation of At4g19440 influences the stability of several mitochondrial RNAs we call this gene MITOCHONDRIAL TRANSCRIPT STABILITY FACTOR 4 (MTSF4). This factor will be an interesting subject to study opposing effects of a single nucleus-encoded protein on mitochondrial transcript levels.