Dataset Information

Cytidine deaminase motifs within the DYW domain of two pentatricopeptide repeat-containing proteins are required for site-specific chloroplast RNA editing.

ABSTRACT: 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.

SUBMITTER: Wagoner JA

PROVIDER: S-EPMC4317000 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Cytidine deaminase motifs within the DYW domain of two pentatricopeptide repeat-containing proteins are required for site-specific chloroplast RNA editing.

Wagoner Jessica A JA Sun Tao T Lin Lin L Hanson Maureen R MR

The Journal of biological chemistry 20141215 5

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, ...[more]

PMID: 25512379

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Project description:The RNA editing occurring in plant organellar genomes mainly involves the change of cytidine to uridine. This process involves a deamination reaction, with cytidine deaminase as the catalyst. Pentatricopeptide repeat (PPR) proteins with a C-terminal DYW domain are reportedly associated with cytidine deamination, similar to members of the deaminase superfamily. PPR genes are involved in many cellular functions and biological processes including fertility restoration to cytoplasmic male sterility (CMS) in plants. In this study, we identified 227 and 211 DYW deaminase-coding PPR genes for the cultivated tetraploid cotton species G. hirsutum and G. barbadense (2n = 4x = 52), respectively, as well as 126 and 97 DYW deaminase-coding PPR genes in the ancestral diploid species G. raimondii and G. arboreum (2n = 26), respectively. The 227 G. hirsutum PPR genes were predicted to encode 52-2016 amino acids, 203 of which were mapped onto 26 chromosomes. Most DYW deaminase genes lacked introns, and their proteins were predicted to target the mitochondria or chloroplasts. Additionally, the DYW domain differed from the complete DYW deaminase domain, which contained part of the E domain and the entire E+ domain. The types and number of DYW tripeptides may have been influenced by evolutionary processes, with some tripeptides being lost. Furthermore, a gene ontology analysis revealed that DYW deaminase functions were mainly related to binding as well as hydrolase and transferase activities. The G. hirsutum DYW deaminase expression profiles varied among different cotton tissues and developmental stages, and no differentially expressed DYW deaminase-coding PPRs were directly associated with the male sterility and restoration in the CMS-D2 system. Our current study provides an important piece of information regarding the structural and evolutionary characteristics of Gossypium DYW-containing PPR genes coding for deaminases and will be useful for characterizing the DYW deaminase gene family in cotton biology and breeding.

Project description:BackgroundThe chloroplast is the organelle responsible for photosynthesis in higher plants. The generation of functional chloroplasts depends on the precise coordination of gene expression in the nucleus and chloroplasts and is essential for the development of plants. However, little is known about nuclear-plastid regulatory mechanisms at the early stage of chloroplast generation in rice.ResultsIn this study, we identified a rice (Oryza sativa) mutant that exhibited albino and seedling-lethal phenotypes and named it ssa1(seedling stage albino1). Transmission electron microscopy (TEM) analysis indicated that the chloroplasts of ssa1 did not have organized thylakoid lamellae and that the chloroplast structure was destroyed. Genetic analysis revealed that the albino phenotypes of ssa1 were controlled by a pair of recessive nuclear genes. Map-based cloning experiments found that SSA1 encoded a pentapeptide repeat (PPR) protein that was allelic to OSOTP51,which was previously reported to participate in Photosystem I (PSI) assembly. The albino phenotype was reversed to the wild type (WT) phenotype when the normal SSA1 sequence was expressed in ssa1 under the drive of the actin promoter. Knockout experiments further created mutants ssa1-2/1-9, which had a phenotype similar to that of ssa1. SSA1 consisted of 7 pentatricopeptide repeat domains and two C-terminal LAGLIDADG tandem sequence motifs and was located in the chloroplast. GUS staining and qRT-PCR analysis showed that SSA1 was mainly expressed in young leaves and stems. In the ssa1 mutants, plastid genes transcribed by plastid-encoded RNA polymerase decreased, while those transcribed by nuclear-encoded RNA polymerase increased at the mRNA level. Loss-of-function SSA1 destroys RNA editing of ndhB-737 and intron splicing of atpF and ycf3-2 in the plastid genome. Yeast two-hybrid and BiFC assays revealed that SSA1 physically interacted with two new RNA editing partners, OsMORF8 and OsTRXz, which have potential functions in RNA editing and chloroplast biogenesis.ConclusionsRice SSA1 encodes a pentatricopeptide repeat protein, which is targeted to the chloroplast. SSA1 regulates early chloroplast development and plays a critical role in RNA editing and intron splicing in rice. These data will facilitate efforts to further elucidate the molecular mechanism of chloroplast biogenesis.

Dataset Information

Cytidine deaminase motifs within the DYW domain of two pentatricopeptide repeat-containing proteins are required for site-specific chloroplast RNA editing.

Publications

Cytidine deaminase motifs within the DYW domain of two pentatricopeptide repeat-containing proteins are required for site-specific chloroplast RNA editing.

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