RNA Editing of Wheat Mitochondrial ATP Synthase Subunit 9: Direct Protein and cDNA Sequencing

Bégu, Dominique; Graves, Pierre‐Vincent; Domec, Christine; Arselin, Geneviève; Litvak, Simón; Araya, Alejandro

doi:10.2307/3869346

Cited by 16 publications

(26 citation statements)

References 17 publications

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“…These results and the previous finding of partial and overedited forms of atp9 mRNA (16) confirm that this process is a posttranscriptional event. The requirements of the wheat mitochondrial lysate in vitro assay show some differences from those of a nuclear lysate system as described for the editing of rat liver apolipoprotein B mRNA (9).…”

Section: Discussionsupporting

confidence: 88%

“…We showed by cDNA sequencing that eight C-to-U changes occurred in wheat mitochondrial atp9 mRNA (15,16). Minor forms of atp9 cDNA with partial or overedited sequences were also found.…”

mentioning

confidence: 93%

“…T. aestivum atp9 cDNA synthesis of lysate-treated RNA and polymerase chain reaction (PCR) were performed as described (16). The 50-du PCR mixture contained 10 mM Tris HCI (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 200 ,uM each dATP, dTTP, dGTP, and dCTP, 50 pmol (1 pAM) of each primer, and 1.2 units of Taq DNA polymerase (Promega).…”

mentioning

confidence: 99%

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An in vitro system for the editing of ATP synthase subunit 9 mRNA using wheat mitochondrial extracts.

Araya¹,

Domec²,

Bégu³

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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A posttranscriptional modification (C-to-U) at specific positions of plant mitochondrial mRNA leads to changes in the amino acid sequence as well as to the emergence of novel initiation or termination sites. This phenomenon, named RNA editing, has been described for several mitochondrial genes from different plant sources. We have found recently that RNA editing of the ATP synthase subunit 9 (aVp9) mRNA involves eight changes including the creation of a new stop codon. In this article, we describe an in vro system devised to follow the editing of wheat mitochondrial a4p9mRNA. Nonedited mRNA was obtained to serve as substrate for this reaction by in vitro transcription of the corresponding gene with T7 RNA polymerase. The source of conversion factor(s) was a soluble fraction obtained from purified wheat mitochondria lysed with salt and detergent. Edited RNA molecules were detected by hybridization with an end-labeled synthetic oligodeoxynucleotide probe complementary to a short region containing four editing events. Optimal conditions for the in viro RNA editing reaction were determined. The reaction is sensitive to high temperature and protease digestion. Pretreatment with micrococcal nuclease decreased RNA editing activity in the mitochondrial extract, suggesting that a nucleic acid is necessary for the enzymatic reactions. Analysis of the edited mRNA showed that the in vitro reaction led to the same products as those observed in vivo.Until recently the protein-coding sequence of a mRNA appeared to be inviolable. However, recent data show that in several instances disparate types of RNA modification can change the coding capacity of mRNA (1). Thus, the mitochondrial genes of a variety of plants undergo posttranscriptional changes at multiple sites creating a U residue in the RNA where a C was found in the genome (2-4). Editing of plant mitochondrial RNA alters the coding sequence in mRNAs, leading to amino acid changes as well as to the emergence of termination or initiation codons (5). Plant mitochondrial RNA editing seems to be somewhat simpler than the phenomenon observed in trypanosome mitochondria (6) but is remarkably similar to the editing of a single residue in the apolipoprotein B mRNA observed in some animal tissues (7, 8). The mechanism by which the C-to-U change operates in plant mitochondria is unknown, although an in vitro system has been described in the case of apolipoprotein B mRNA modification (9). A model has been proposed for RNA editing in kinetoplastid mitochondria involving "guide" RNA molecules transcribed from maxicircle or minicircle mitochondrial DNA (10).In studying the organization and expression of protein genes essential for wheat mitochondrial function, our laboratory has mapped and sequenced several wheat mitochondrial genes (11)(12)(13)(14). We showed by cDNA sequencing that eight C-to-U changes occurred in wheat mitochondrial atp9 mRNA (15, 16). Minor forms of atp9 cDNA with partial or overedited sequences were also found. Partial protein sequencing showed that edit...

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Section: Discussionsupporting

confidence: 88%

“…We showed by cDNA sequencing that eight C-to-U changes occurred in wheat mitochondrial atp9 mRNA (15,16). Minor forms of atp9 cDNA with partial or overedited sequences were also found.…”

mentioning

confidence: 93%

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An in vitro system for the editing of ATP synthase subunit 9 mRNA using wheat mitochondrial extracts.

Araya¹,

Domec²,

Bégu³

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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“…From a fundamental point of view, these results reinforce the idea that RNA editing is required for the synthesis of functionally competent proteins (26,27). Although the experimental model used in this study seems attractive to test the functionality of unedited protein products, it should be remembered that all the proteins encoded in mitochondria do not have the same importance in the physiology of the organelle.…”

Section: Ms Asupporting

confidence: 75%

Transgenic male-sterile plant induced by an unedited atp9 gene is restored to fertility by inhibiting its expression with antisense RNA.

Zabaleta

Mouras

Hernould

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

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We have previously shown that the expression of an unedited atp9 chimeric gene correlated with malesterile phenotype in transgenic tobacco plant. To study the relationship between the expression of chimeric gene and the male-sterile trait, hemizygous and homozygous transgenic tobacco lines expressing the antisense atp9 RNA were constructed. The antisense producing plants were crossed with a homozygous male-sterile line, and the Fl progeny was analyzed. The offspring from crosses between homozygous lines produced only male-fertile plants, suggesting that the expression antisense atp9 RNA abolishes the effect of the unedited chimeric gene. In fact, the plants restored to male fertility showed a dramatic reduction of the unedited atp9 transcript levels, resulting in normal flower development and seed production. These results support our previous observation that the expression of unedited atp9 gene can induce male sterility.The development of strategies to improve crop plants by the production of hybrid varieties is a major goal in plant breeding. Hybrid progeny often have a higher yield, increased resistance to disease, and an enhanced performance in different environments compared with the parental lines (1). Male sterility mutations that guarantee the outcrossing of naturally autogamic plant lines (2) have proven to be useful for the production of hybrid lines with increased crop productivity. However, hybrid production is limited to those species of plants in which male sterility mutations have been detected. Moreover, in certain plants, such as corn, wheat, rice, or tomato, where seeds and fruits are harvested products, a male fertility restorer system is required.Many male sterility mutations interfere with tapetal cell differentiation and/or function, indicating that this tissue is essential for the production of functional pollen grains (3-7).The tapetal cells are the target to produce engineered malesterile plants. In view of this, several authors have reported the production of transgenic male-sterile plants by the expression of degradative enzymes in tapetal cells (8)(9)(10) or by the inhibition of particular enzymes by antisense strategies (11). One interesting, naturally-occurring phenotype is cytoplasmic male sterility. Molecular analysis of cytoplasmic male sterility plants has shown the presence of chimeric genes arising from rearrangement of the mtDNA. The production of proteins encoded by the chimeric genes has been correlated with male-sterile phenotype, and it has been proposed that it affects mitochondrial function (12-14).The different nature of the genes involved in cytoplasmic male sterility plants reported so far suggests that mitochondrial impairment may be the common consequence of the different chimeric gene products. If this is true, then a mitochondrial dysfunction could also be produced by other means and male-sterile plants could be obtained. Recently, we have reported the production of male-sterile tobacco plants by expression of the unedited coding region of mitochondrial ...

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“…It is an important process during gene expression in plant mitochondria since the organelle encoded proteins are issued *Corresponding author. Fax: (33) (56) 99-9057. from edited mRNAs [10]. RNA editing occurs also in some chloroplast transcripts although at a lesser extent than in mitochondria [11].…”

Section: Introductionmentioning

confidence: 99%

RNA editing in wheat mitochondria proceeds by a deamination mechanism

1995

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RNA Editing of Wheat Mitochondrial ATP Synthase Subunit 9: Direct Protein and cDNA Sequencing

Cited by 16 publications

References 17 publications

An in vitro system for the editing of ATP synthase subunit 9 mRNA using wheat mitochondrial extracts.

An in vitro system for the editing of ATP synthase subunit 9 mRNA using wheat mitochondrial extracts.

Transgenic male-sterile plant induced by an unedited atp9 gene is restored to fertility by inhibiting its expression with antisense RNA.

RNA editing in wheat mitochondria proceeds by a deamination mechanism

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