A DYW domain-containing pentatricopeptide repeat protein is required for RNA editing at multiple sites in mitochondria of Arabidopsis thaliana

Abstract

RNA editing in flowering plant mitochondria alters 400 to 500 nucleotides from C to U, changing the information content of most mRNAs and some tRNAs. So far, none of the specific or general factors responsible for RNA editing in plant mitochondria have been identified. Here, we characterize a nuclear-encoded gene that is involved in RNA editing of three specific sites in different mitochondrial mRNAs in Arabidopsis thaliana, editing sites rps4-956, nad7-963, and nad2-1160. The encoded protein MITOCHONDRIAL RNA EDITING FACTOR1 (MEF1) belongs to the DYW subfamily of pentatricopeptide repeat proteins. Amino acid identities altered in MEF1 from ecotype C24, in comparison to Columbia, lower the activity at these editing sites; single amino acid changes in mutant plants inactivate RNA editing. These variations most likely modify the affinity of the editing factor to the affected editing sites in C24 and in the mutant plants. Since lowered and even absent RNA editing is tolerated at these sites, the amino acid changes may be silent for the respective protein functions. Possibly more than these three identified editing sites are addressed by this first factor identified for RNA editing in plant mitochondria.

Figure 1.

The MEF1 Nuclear Gene Is Responsible for Ecotype-Specific RNA Editing Variations in Arabidopsis Mitochondria. (A) Ecotype C24 shows partial RNA editing at the mitochondrial editing sites rps4-956 and nad7-963 where both Ler and Col ecotypes are fully edited from C to U. At site nad2-1160, the RNA editing level in ecotype C24 is only slightly lower than the 100% in Col and Ler. (B) Introduction of the Col version of gene MEF1 (35S:MEF1 sequence traces) into C24 protoplasts increases RNA editing at sites rps4-956 and nad7-963 to levels higher than those of untransfected C24 protoplasts (top traces). Sequence traces of wild-type ecotype Col (bottom sequence traces) and of C24 protoplasts transfected with GFP (35S:GFP) as a control are also shown. Other editing sites in the rps4 mRNA are not affected.

Figure 2.

RNA Editing Analysis of Two Ecotype Col-Derived Plant Lines Mutated in the Nuclear Locus MEF1. Two plant lines with independent mutations (mef1-1 and mef1-2) in the MEF1 gene show no editing at the rps4-956 site in leaves or in protoplasts (top panels, control). Transfection of protoplasts from mutants mef1-1 and mef1-2 with the wild-type Col MEF1 gene restores the ability to edit this site in the rps4 mRNAs. Introduction of the gene for GFP does not influence editing. The two mutants are also defective in editing at site nad7-963 (center panels, control). At the third site nad2-1160, RNA editing is lowered but still detectable (bottom panels, control). Transfection of protoplasts from the mutants mef1-1 and mef1-2 with the wild-type Col MEF1 gene restores the ability to edit site nad7-963 and enhances editing at nad2-1160. The MEF1 gene is thus required for, or at least involved in, RNA editing at these three sites in mitochondria of Arabidopsis.

Figure 3.

Transcript Patterns of the Mitochondrial Target RNAs in Col, C24, and the MEF1 Mutants. The transcript patterns of the mitochondrial nad2, nad7, and rps4 genes are compared by RNA gel blot analysis. The hybridization signals of the gene-specific probes show comparable patterns and similar amounts of the respective precursors and mature transcripts from all three genes in Col, C24, and the mef1-1 and mef1-2 mutants. The specificity of the probes is demonstrated by dot blot hybridization to the three gene sequences shown underneath each panel, with the order of the dots being nad2, nad7, and rps4 from left to right on each filter. The source of the respective total cellular RNA preparation is given above each lane. The positions of DNA size standards are indicated alongside in kilobase pairs.

Figure 4.

Analysis of the MEF1 Transcript Abundance in Col Wild-Type and Mutant Plants. The relative amounts of MEF1 transcripts are analyzed by reverse transcription and subsequent cycle-limited PCR in Col and in the mef1-1 and mef1-2 mutants. The gel pictures show the transcript signals generated after the indicated number of cycles. Transcripts of another unrelated PPR gene (At1g59720) were amplified in parallel as a standard for the amount of RNA and as a PCR control.

Figure 5.

Structure of the DYW-PPR-Class MEF1 Protein and Locations of the Ecotype Variations and the mef1-1 and mef1-2 Mutations. (A) The order of the PPR repeats and the E/E+/DYW domains are displayed in this protein schematic with the locations of the changes caused by the various SNPs (arrows). The labeling of the PPR repeats follows the nomenclature introduced by Lurin et al. (2004). (B) The deduced amino acid sequence of MEF1 is shown. The three nonsilent variations between C24 and Col, the single amino acid change between Col and Ler, and the single mutations in mef1-1 and mef1-2 are indicated. Residues conserved according to Lurin et al. (2004) are in bold. The motifs in which the changes are located are boxed and their classifications are given. The effected amino acid changes are indicated above the protein sequence.

Figure 6.

Alignment of the Putative cis-Specificity Regions around the RNA Editing Sites Targeted by MEF1. Editing at the first two sites, rps4-956 and nad7-963, and to a lesser extent also at the third, nad2-1160, is influenced by the mef1-1 and mef1-2 mutations and the C24/Col variations. The edited C is in bold and a larger font. Five further nucleotide identities shared between the three sites are inversely shaded. Two other editing sites, ccb203-65 and ccb382-955, share the same five nucleotides, but their editing is not affected in C24 or in the mutant mef1-2. Editing sites rps4-956 and nad2-1160 alter amino acid codons, both changing S→L, while editing site nad7-963 is silent. The two unaffected editing sites, ccb203-65 and ccb382-955, both alter codons, the first also S→ L and the latter R→C.