Plastid signalling under multiple conditions is accompanied by a common defect in RNA editing in plastids

Retrograde signalling from the plastid to the nucleus, also known as plastid signalling, plays a key role in coordinating nuclear gene expression with the functional state of plastids. Inhibitors that cause plastid dysfunction have been suggested to generate specific plastid signals related to their modes of action. However, the molecules involved in plastid signalling remain to be identified. Genetic studies indicate that the plastid-localized pentatricopeptide repeat protein GUN1 mediates signalling under several plastid signalling-related conditions. To elucidate further the nature of plastid signals, investigations were carried out to determine whether different plastid signal-inducing treatments had similar effects on plastids and on nuclear gene expression. It is demonstrated that norflurazon and lincomycin treatments and the plastid protein import2-2 (ppi2-2) mutation, which causes a defect in plastid protein import, all resulted in similar changes at the gene expression level. Furthermore, it was observed that these three treatments resulted in defective RNA editing in plastids. This defect in RNA editing was not a secondary effect of down-regulation of pentatricopeptide repeat protein gene expression in the nucleus. The results indicate that these three treatments, which are known to induce plastid signals, affect RNA editing in plastids, suggesting an unprecedented link between plastid signalling and RNA editing.     

The E-class PPR protein MEF3 of Arabidopsis thaliana can also function in mitochondrial RNA editing with an additional DYW domain

In plants, RNA editing is observed in mitochondria and plastids, changing selected C nucleotides into Us in both organelles. We here identify the PPR (pentatricopeptide repeat) protein MEF3 (mitochondrial editing factor 3) of the E domain PPR subclass by genetic mapping of a variation between ecotypes Columbia (Col) and Landsberg erecta (Ler) in Arabidopsis thaliana to be required for a specific RNA editing event in mitochondria. The Ler variant of MEF3 differs from Col in two amino acids in repeats 9 and 10, which reduce RNA editing levels at site atp4-89 to about 50% in Ler. In a T-DNA insertion line, editing at this site is completely lost. In Vitis vinifera the gene most similar to MEF3 continues into a DYW extension beyond the common E domain. Complementation assays with various combinations of PPR and E domains from the vine and A. thaliana proteins show that the vine E region can substitute for the A. thaliana E region with or without the DYW domain. These findings suggest that the additional DYW domain does not disturb the MEF3 protein function in mitochondrial RNA editing in A. thaliana.     

Cytidine Deaminase Motifs within the DYW Domain of Two Pentatricopeptide Repeat-containing Proteins Are Required for Site-specific Chloroplast RNA Editing

In angiosperm organelles, cytidines are converted to uridines by a deamination reaction in the process termed RNA editing. The C targets of editing are recognized by members of the pentatricopeptide repeat (PPR) protein family. Although other members of the editosome have begun to be identified, the enzyme that catalyzes the C-U conversion is still unknown. The DYW motif at the C terminus of many PPR editing factors contains residues conserved with known cytidine deaminase active sites; however, some PPR editing factors lack a DYW motif. Furthermore, in many PPR-DYW editing factors, the truncation of the DYW motif does not affect editing efficiency, so the role of the DYW motif in RNA editing is unclear. Here, a chloroplast PPR-DYW editing factor, quintuple editing factor 1 (QED1), was shown to affect five different plastid editing sites, the greatest number of chloroplast C targets known to be affected by a single PPR protein. Loss of editing at the five sites resulted in stunted growth and accumulation of apparent photodamage. Adding a C-terminal protein tag to QED1 was found to severely inhibit editing function. QED1 and RARE1, another plastid PPR-DYW editing factor, were discovered to require their DYW motifs for efficient editing. To identify specific residues critical for editing, conserved deaminase residues in each PPR protein were mutagenized. The mutant PPR proteins, when expressed in qed1 or rare1 mutant protoplasts, could not complement the editing defect. Therefore, the DYW motif, and specifically, the deaminase residues, of QED1 and RARE1 are required for editing efficiency.     

Predictable Alteration of Sequence Recognition by RNA Editing Factors from Arabidopsis

RNA editing factors of the pentatricopeptide repeat (PPR) family show a very high degree of sequence specificity in the recognition of their target sites. A molecular basis for target recognition by editing factors has been proposed based on statistical correlations but has not been tested experimentally. To achieve this, we systematically mutated the pentatricopeptide motifs in the Arabidopsis thaliana RNA editing factor CLB19 to investigate their individual contribution to RNA recognition. We find that the motifs contributing significantly to the specificity of binding follow the previously proposed recognition rules, distinguishing primarily between purines and pyrimidines. Our results are consistent with proposals that each motif recognizes one nucleotide in the RNA target with the protein aligned parallel to the RNA and contiguous motifs aligned with contiguous nucleotides such that the final PPR motif aligns four nucleotides upstream of the edited cytidine. By altering S motifs in CLB19 and another editing factor, OTP82, and using the modified proteins to attempt to complement the respective mutants, we demonstrate that we can predictably alter the specificity of these factors in vivo.