Identification of the chloroplast adenosine-to-inosine tRNA editing enzyme - PubMed (original) (raw)

Identification of the chloroplast adenosine-to-inosine tRNA editing enzyme

Daniel Karcher et al. RNA. 2009 Jul.

Abstract

Plastids (chloroplasts) of higher plants exhibit two types of conversional RNA editing: cytidine-to-uridine editing in mRNAs and adenosine-to-inosine editing in at least one plastid genome-encoded tRNA, the tRNA-Arg(ACG). The enzymes catalyzing RNA editing reactions in plastids are unknown. Here we report the identification of the A-to-I tRNA editing enzyme from chloroplasts of the model plant Arabidopsis thaliana. The protein (AtTadA) has an unusual structure in that it harbors a large N-terminal domain of >1000 amino acids, which is not required for catalytic activity. The C-terminal region of the protein displays sequence similarity to tadA, the tRNA adenosine deaminase from Escherichia coli. We show that AtTadA is imported into chloroplasts in vivo and demonstrate that the in vitro translated protein triggers A-to-I editing in the anticodon of the plastid tRNA-Arg(ACG). Suppression of AtTadA gene expression in transgenic Arabidopsis plants by RNAi results in reduced A-to-I editing in the chloroplast tRNA-Arg(ACG). The RNAi lines display a mild growth phenotype, presumably due to reduced chloroplast translational efficiency upon limited availability of edited tRNA-Arg(ACG).

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Figures

FIGURE 1.

FIGURE 1.

Partial sequence alignment of the tadA protein from Escherichia coli (E.c. tadA) and the candidate AtTadA protein from Arabidopsis thaliana (At1g68720). Identical amino acid residues are shown in red, similar amino acid residues in blue. Amino acids involved in zinc binding are underlined. Note that At1g68720 has a large N-terminal extension of >1000 amino acids, which has no sequence similarity to any protein of known function and is not shown here.

FIGURE 2.

FIGURE 2.

Analysis of subcellular localization of the candidate AtTadA protein from Arabidopsis thaliana. A chimeric protein consisting of the first 177 amino acids of At1g68720 fused to GFP is targeted to chloroplasts in transformed tobacco protoplasts. Fluorescence of the green fluorescence protein (GFP), chlorophyll fluorescence (Chl), and the overlay of the two fluorescences are shown for two transformed protoplasts.

FIGURE 3.

FIGURE 3.

Analysis of tRNA editing in transgenic Arabidopsis plants harboring anRNAi construct targeted against the candidate AtTadA gene, At1g68720. (A) Down-regulation of At1g68720 expression in three independently generated RNAi lines (RNAi-_tadA_-E, RNAi-_tadA_-F, and RNAi-_tadA_-H). RT-PCR data comprise three biological replicas. Error bars indicate standard deviation. (B) Analysis of A-to-I editing in the anticodon of the chloroplast genome-encoded tRNA-Arg(ACG). Note that the sequence chromatographs show the sequence of the complementary strand and, therefore, A-to-I conversion is seen as T-to-C conversion here. While the wild type (Wt) shows nearly complete A-to-I editing (C peak marked by arrow), the RNAi lines show drastically reduced editing, the extent of which correlates with the level of downregulation of At1g68720 by RNAi (A).

FIGURE 4.

FIGURE 4.

Phenotype of RNAi–tadA plants. Wild-type (Wt) and mutant plants were grown under long-day conditions at a light intensity of 180 μE m−2 sec−1. All RNAi lines show a mild growth retardation as evidenced by delayed flowering. The delayed growth correlates with the level of down-regulation of At1g68720 and the concomitant reduction in tRNA editing efficiency in the individual RNAi lines (Fig. 3).

FIGURE 5.

FIGURE 5.

A-to-I RNA editing activity of the chloroplast AtTadA protein in vitro. Largely unedited tRNA-Arg(ACG) from an RNAi-_tadA_-F plant was used as substrate for in vitro editing assays with the chloroplast AtTadA protein produced by in vitro translation. As a control, GFP was produced by in vitro translation. While incubation with the AtTadA protein leads to time-dependent A-to-I conversion in the anticodon of tRNA-Arg(ACG), the tRNA remains largely unedited in the GFP control. The chromatographs show the sequence of the complementary strand. A-to-I conversion is, therefore, seen as T-to-C conversion here.

References

    1. Bass BL. RNA editing and hypermutation by adenosine deamination. Trends Biochem Sci. 1997;22:157–162. - PubMed
    1. Bock R. Sense from nonsense: How the genetic information of chloroplasts is altered by RNA editing. Biochimie. 2000;82:549–557. - PubMed
    1. Bock R. RNA editing in plant mitochondria and chloroplasts. In: Bass B, editor. Frontiers in molecular biology: RNA editing. Oxford University Press; New York: 2001. pp. 38–60.
    1. Bock R, Koop H-U. Extraplastidic site-specific factors mediate RNA editing in chloroplasts. EMBO J. 1997;16:3282–3288. - PMC - PubMed
    1. Bock R, Kössel H, Maliga P. Introduction of a heterologous editing site into the tobacco plastid genome: The lack of RNA editing leads to a mutant phenotype. EMBO J. 1994;13:4623–4628. - PMC - PubMed

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