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

Abstract

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...

RNA editing in wheat mitochondria proceeds by a deamination mechanism

“…Our previous preliminary results showed that treatment of the wheat mitochondrial lysate with micrococcal nuclease partially reduced the editing activity [13] suggesting the presence of a nucleic acid factor involved in plant mitochondrial editing. When we addressed this question with the TLC of P1 digestion assay (Fig.…”

“…For this purpose an in vitro assay system using a labelled probed complementary to the edited form of a region of atp9 mRNA containing 4 editing events was developed [13]. To understand the mechanism of the editing reaction, we used a construction carrying the unedited atp9 coding region ( Fig.…”

“…To understand the mechanism of RNA editing in plant mitochondria, the unedited RNA probe was incubated with the lysate prepared as in [13] or with fractions obtained by chromatography on MonoQ column. After one or two hour incubation, no digestion of the RNA probe was observed, suggesting that no endonucleolytic activity is detected in the extract (results not shown).…”

“…Labelled substrate was incubated with protein fractions as described [13]. P1 nuclease digestion of atp9 RNA was performed after phenol:chloroform extraction of the RNA editing incubation mixtures.…”

“…After 2 h incubation at room temperature, the reaction mixture was made 1 mM in EGTA. Four/11 of the MN treated proteins were used for the editing reaction in the conditions described [13]. Controls made without MN and without EGTA during 0014-5793/95/$9.50 © 1995 Federation of European Biochemical Societies.…”