Distinct role of Arabidopsis mitochondrial P-type pentatricopeptide repeat protein-modulating editing protein, PPME, in nad1 RNA editing

Abstract

The mitochondrion is an important power generator in most eukaryotic cells. To preserve its function, many essential nuclear-encoded factors play specific roles in mitochondrial RNA metabolic processes, including RNA editing. RNA editing consists of post-transcriptional deamination, which alters specific nucleotides in transcripts to mediate gene expression. In plant cells, many pentatricopeptide repeat proteins (PPRs) participate in diverse organellar RNA metabolic processes, but only PLS-type PPRs are involved in RNA editing. Here, we report a P-type PPR protein from Arabidopsis thaliana, P-type PPR-Modulating Editing (PPME), which has a distinct role in mitochondrial nad1 RNA editing via RNA binding activity. In the homozygous ppme mutant, cytosine (C)-to-uracil (U) conversions at both the nad1-898 and 937 sites were abolished, disrupting Arg(300)-to-Trp(300) and Pro(313)-to-Ser(313) amino acid changes in the mitochondrial NAD1 protein. NAD1 is a critical component of mitochondrial respiration complex I; its activity is severely reduced in the homozygous ppme mutant, resulting in significantly altered growth and development. Both abolished RNA editing and defective complex I activity were completely rescued by CaMV 35S promoter- and PPME native promoter-driven PPME genomic fragments tagged with GFP in a homozygous ppme background. Our experimental results demonstrate a distinct role of a P-type PPR protein, PPME, in RNA editing in plant organelles.

Keywords: Arabidopsis; PPR; complex I activity; editing; mitochondria; nad1.

Figure 1.

P-type pentatricopeptide repeat protein-modulating editing (PPME) protein is a P-type pentatricopeptide repeat (PPR) protein essential for normal Arabidopsis growth and development. (A) The PPME gene structure and its encoded P-type PPR protein harboring 16 PPR (35 amino acids) motifs is shown; ppme-1 was generated by T-DNA insertion in the coding region. (B) RT-PCR analysis of constitutive PPME expression, of a null homozygous ppme-1 mutant and of transformed PPME in T3 transgenic lines complemented with CaMV 35S promoter-driven (35S::PPME-GFP/ppme−/−, comp1) or PPME native promoter-driven (PPMEg-GFP/ppme−/−, comp2) PPME genomic fragments in a homozygous ppme-1 background. (C) Fourteen-day-old heterozygous ppme+/− germinated F2 seedlings grown on solid MS medium. (D) Fourteen-day-old homozygous ppme−/− seedlings with stunted vegetative growth. (E) Forty-day-old homozygous ppme−/− plants survived on solid MS medium for 21 d before being transferred to soil. (F-G) Fourteen-day-old seedlings (F) and 40-day-old (G) plants from wild-type, homozygous ppme−/−, comp1, and comp2 plants. (H-I) Siliques of homozygous ppme−/− showed an abortion phenotype (H), with shrunken seeds and reduced viability (I). Scale bars = 0.5 cm in C-G; 2 mm in H; and 500 μm in I.

Figure 2.

Specific localization of PPME in Arabidopsis plant mitochondria. Root hairs from 7-day-old PPMEg-GFP transgenic plants were treated with Mito-tracker to observe PPME subcellular localization. Co-localization of the GFP (green) and Mito-tracker signals (red) in elongating root hair cells, as observed by confocal microscopy. Scale bars = 10 μm.

Figure 3.

Defective editing at mitochondrial nad1-898 and nad1-937 RNA editing sites in homozygous ppme−/− seedlings. (A) Sequencing of cDNAs from 14-day-old wild-type, homozygous ppme−/−, and complemented seedlings for assessment of nad1-898 and nad1-937 RNA editing efficiencies in ppme−/− and complementation lines. The middle cytosine or thymine in each panel is the position of the nad1-898 or nad1-937 site, respectively. The lower panel depicts the predicted amino acid changes after nad1-898 and nad1-937 editing. In wild-type mitochondria, nad1-898 and nad1-937 RNA editing causes amino acid changes from Arg to Trp and from Pro to Ser, respectively, in the NAD1 protein after translation. The bold cytosine (C) and uracil (U) indicate the nad1-898 and nad1-937 editing sites, respectively. (B) Poisoned primer extension assay of the nad1-898 editing site. The edited products were terminated earlier than the unedited products by stopping the reaction with ddATP. The edited and unedited products were separated in a sequencing gel and visualized by detection of FAM fluorescence signals. The edited products are from wild-type and ppme−/− seedlings and seedlings from 2 complementation lines.

Figure 4.

RNA-EMSA showing that recombinant PPME specifically binds to sequences surrounding the nad1-898 editing site. (A) MBP-tagged PPME recombinant proteins were co-incubated with nad1-898 probes or atp9-83 probes for sequences located up- and downstream of nad1-898 or atp9-83, respectively. The left panel shows the interaction between MBP-PPME and nad1-898 or atp9-83. The black triangles above each gel indicate the increasing concentrations of MBP-PPME in each gel. The right panel shows the binding between MBP-PPME and nad1-898, which was titrated by the exogenous addition of cold nad1-898 probe. +: cold competitor. (B) Nucleotide sequences of the probes specifically designed for EMSA. The RNA sequence (from 5′ to 3′) includes the region from the -40 nucleotide of nad1-898 to the +1 nucleotide of nad1-937. The bold C nucleotides indicate the corresponding nad1-898, nad1-928, and nad1-937 editing sites. The bold solid lines indicate the regions individually probed with specific probes, and the dotted line represents the putative nad1-898 cis-element recognized by PPME. . (C) RNA-EMSA revealed that among the 4 different probes, PPME specifically bound to only the nad1-898 −20 to +1 and nad1-928 -30 to +1 probes. However, the 20 nucleotides (putative cis-element for nad1-928) upstream of nad1-928 that overlapped with the nad1-937 −30 to +1 region did not exhibit PPME binding activity. The atp9-83 −20 to +1 probe was used as a cis-element negative control. The + and ++ symbols denote 200 nM MBP and 100 nM MBP-PPME or 200 nM MBP-PPME recombinant protein, respectively, and 200 pM probe was used for the all RNA-EMSAs.

Figure 5.

Accurate nad1-898 and nad1-937 editing plays an important role in Arabidopsis mitochondrial complex I activity. (A) Comparison of the amino acid identities of NAD1 C-termini from different organisms shows that the conserved edited form of nad1-898 has an amino acid change to Trp and that the less-conserved edited form of nad1-937 has an amino acid change to Ser. The black arrows from the left to right indicate the amino acids translated from the nad1-898 and nad1-937 editing sites, respectively. (B) The significant reduction in mitochondrial complex I activity in the ppme mutant was restored in the complementation lines. Crude total mitochondrial protein extracts from 14-day-old seedlings were separated by native PAGE. The activity was visualized as a purple-blue color resulting from interaction of the substrate (NADH) with the electron acceptor (nitroblue tetrazolium). The left panel shows the total protein profiles, which were determined by silver staining, and the right panel shows the parallel native PAGE staining for assessment of mitochondrial complex I activity. The black arrow shows mitochondrial complex I.