RNA PROCESSING FACTOR 5 is required for efficient 5′ cleavage at a processing site conserved in RNAs of three different mitochondrial genes in Arabidopsis thaliana

Abstract

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.

RNA Processing Factor 7 and Polynucleotide Phosphorylase Are Necessary for Processing and Stability of nad2 mRNA in Arabidopsis Mitochondria

“…Previous studies of RPF5 required for 5′ maturation of 26S rRNA as well as nad6 and atp9 mRNAs, revealed that the knockout of this gene leads to an accumulation of nad6 precursor RNAs while both mature and 5′ un-processed 26S rRNA are substantially reduced. 33 This observation indicated that the 5′ extended 26S rRNA precursors are less stable than the 5′ mature RNA species suggesting that 5′ processing is a prerequisite for adequate accumulation of the mature 26S rRNA species. In plant mitochondria, PNPase, a 3′ to 5′ exoribonuclease, is involved in the generation of mature 3′ termini of transcripts such as atp9 mRNA but is also required for the removal of RNAs from the total mitochondrial transcript pool.…”

“…These factors, exclusively P-class PPR proteins comprising 14 PPR motifs or more, are required for efficient 5′ processing of the major transcript species transcribed from one, two or three different mitochondrial genes, [25][26][27]33 but as found recently by the analysis of the restorer of fertility-like PPR protein 9 (RFL9), are also involved in 5′ processing of rare RNA molecules in particular ecotypes. 38 The relatively high number of PPR motifs present in these proteins allowed to predict the putative binding sites on the RNA targets, which is exclusively found upstream of the 5′ end of the mature transcript.…”

“…This observation suggests that severely reduced 5′ processing of nad2 transcripts interferes with the stability of these RNA similar to what have been observed for 26S rRNA. 33 These observations raise the possibility that 5′ processing somehow interferes with transcript stability, but still it remains unclear whether the reduced stability of 5′ un-processed transcripts is a general phenomenon in plant mitochondria. Furthermore, the amplification reaction directed to the nad2 reading frame revealed a total decrease of nad2 transcripts both in rpf7-1 and PNPase knockdown plants (Fig.…”

“…Only the loss of RPF5, a canonical P-class PPR protein involved in processing of nad6 and atp9 mRNAs as well as 26S rRNA interferes with the seed germination. 33 To obtain more information about 5′ processing in plant mitochondria, we now used a rare nad2 mRNA polymorphism in Arabidopsis accession Can-0 to identify the gene encoding RPF7. This protein, required for the efficient processing at the -122 5′ terminus of the nad2 mRNA, is a short P-class member, composed of seven canonical P and a single short S motif.…”

The Rf and Rf-like PPR in higher plants, a fast-evolving subclass of PPR genes

“…6 However, 5'-end processing driven by endonucleolytic cleavage is also associated with mitochondria-targeted PPR proteins that are unrelated to the RFL family, ruling out an exclusive role for RFL proteins in this type of transcript processing. 58,59 As indicated, the activities supported by the three characterized Arabidopsis RFL do not appear to be essential for the expression of their target mitochondrial transcripts, and corresponding rfl mutants do not show any obvious phenotypical alterations compared with wild-type plants. It cannot be ruled out though that the differential processing of mitochondrial mRNA ends could alter unobvious fitness traits, modulating in subtle ways adaptive capacity of the different Arabidopsis accessions harboring distinct alleles of the concerned RFL genes.…”