Importance of adenosine-to-inosine editing adjacent to the anticodon in an Arabidopsis alanine tRNA under environmental stress - PubMed (original) (raw)

. 2013 Mar 1;41(5):3362-72.

doi: 10.1093/nar/gkt013. Epub 2013 Jan 25.

Affiliations

Importance of adenosine-to-inosine editing adjacent to the anticodon in an Arabidopsis alanine tRNA under environmental stress

Wenbin Zhou et al. Nucleic Acids Res. 2013.

Abstract

In all organisms, transfer RNAs (tRNAs) undergo extensive post-transcriptional modifications. Although base modifications in the anticodon are known to alter decoding specificity or improve decoding accuracy, much less is known about the functional relevance of modifications in other positions of tRNAs. Here, we report the identification of an A-to-I tRNA editing enzyme that modifies the tRNA-Ala(AGC) in the model plant Arabidopsis thaliana. The enzyme is homologous to Tad1p, a yeast tRNA-specific adenosine deaminase, and it selectively deaminates the adenosine in the position 3'-adjacent to the anticodon (A37) to inosine. We show that the AtTAD1 protein is exclusively localized in the nucleus. The tad1 loss-of-function mutants isolated in Arabidopsis show normal accumulation of the tRNA-Ala(AGC), suggesting that the loss of the I37 modification does not affect tRNA stability. The tad1 knockout mutants display no discernible phenotype under standard growth conditions, but produce less biomass under environmental stress conditions. Our results provide the first evidence in support of a physiological relevance of the A37-to-I modification in eukaryotes.

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Figures

Figure 1.

Figure 1.

Subcellular localization of a TAD1–GFP fusion protein expressed from the CaMV 35S promoter in stably transformed Arabidopsis plants. Images were obtained by confocal laser-scanning microscopy. Scale bars = 10 µm. (A–C) Localization of the TAD1–GFP fusion protein in hypocotyl cells. (A) GFP fluorescence. (B) Chlorophyll fluorescence. (C) Overlay of GFP and chlorophyll fluorescence. (D–F) Localization of the TAD1–GFP fusion protein in root tips. (D) GFP fluorescence. (E) Bright field image. (C) Overlay of GFP fluorescence and bright field image.

Figure 2.

Figure 2.

Identification and characterization of tad1 mutants in Arabidopsis. (A) Exon–intron structure of the putative TAD1 locus and location of the T-DNA insertions in the tad1-1 and tad1-2 mutants (indicated by open triangles). The arrowheads denote location and orientation of the primers used in (B). (B) Identification of homozygous T-DNA insertions in the tad1 mutants by PCR using genomic DNA as template. Primer combinations are indicated at the right. Two independent samples were analyzed for each plant line. Failure to obtain PCR products with gene-specific primer pairs in the mutants (primers F1 and R1 for tad1-1 and primers F2 and R2 for tad1-2) confirmed homozygosity of the T-DNA insertions in the TAD1 locus. (C) Detection of TAD1 mRNA in the wild-type (WT) and the two tad1 mutants by semiquantitative RT–PCR. The mRNA for an actin isoform (ACTIN2) served as internal RT–PCR control. Note residual TAD1 expression in the tad1-1 mutant, but virtual absence of transcripts from the tad1-2 mutant. (D) Quantitation of TAD1 transcript levels in T-DNA mutants by qRT–PCR. mRNA accumulation in the tad1-1 and tad1-2 mutants is shown relative to the wild-type level (set to 100%). (E) Detection of TAD1 transcripts in various tissues of Arabidopsis wild-type plants by semiquantitative RT–PCR.

Figure 3.

Figure 3.

Analysis of tRNA-Ala(AGC) editing in tad1 mutants. (A) Cloverleaf structure of the tRNA-Ala(AGC) from Arabidopsis. Watson–Crick base pairing and UG base pairing are represented by bars and open circles, respectively. TAD1 deaminates A37 in the anticodon loop of the tRNA. I37 undergoes further modification by _S_-adenosylmethionine–dependent methylation to N1-methylinosine (m1I37; 45). m1I37 is read as T by reverse transcriptases (indicated by the dotted arrow). In yeast, two other deaminase proteins, TAD2 and TAD3, form a heterodimer and specifically deaminate A34 (46). Reverse transcriptases read I as G. (B) Analysis of A-to-I editing at positions 34 and 37 of tRNA-Ala(AGC) in wild-type Arabidopsis plants (WT), the tad1 mutants, tad1-1 and tad1-2, and the complemented tad1-2 line (Compl.). The tRNAs were reverse transcribed, the cDNA sequences amplified by PCR and directly sequenced. Note loss of A-to-I editing at position 37 in tad1 mutants (as evidenced by presence of the genomically encoded A instead of the T originating from reverse transcription of m1I-37 in the wild-type), but unaffected editing at position 34 (G indicating presence of inosine in both the wild-type and the mutants). As a control, two other inosine-containing cytosolic tRNAs (a threonine and a valine tRNA) were also investigated and, likewise, turned out to be unaffected in the Arabidopsis tad1 mutants. DNA indicates genomic DNA; cDNA denotes complementary DNA.

Figure 4.

Figure 4.

Accumulation and aminoacylation of tRNA-Ala(AGC) in wild-type plants (WT), tad1 mutant plants and the complemented tad1-2 line (Compl.). Northern blot analyses were carried out with total cellular RNA (A) or purified mitochondrial RNA (B). To control for loading, the ethidium bromide-stained agarose gels before blotting are also shown. Accumulation of the mitochondrial encoded tRNA-Cys(GCA) was analyzed as a control in (B). The 26S and 18S rRNAs of the mitochondrial ribosome and the tRNA band are indicated in the ethidium bromide-stained agarose gels. To additionally control for loading differences visible in the mitochondrial RNA gel blots, the blots were stripped and re-hybridized to a probe specific to the mitochondrial 5S rRNA. (C) Analysis of aminoacylation of tRNA-Ala(AGC) in the wild-type, the tad1 mutant and the complemented tad1-2 line. In the gel system used here (42), the aminoacylated tRNA migrates slower than its corresponding deacylated species. To visualize the difference in electrophoretic mobility, aliquots of the wild-type sample and the tad1-2 sample were deacylated in vitro. Samples of 8 µg of RNA were separated by electrophoresis, blotted and hybridized to a tRNA-Ala(AGC)-specific probe.

Figure 5.

Figure 5.

Phenotype of tad1 mutant plants in comparison with the wild-type (WT) and the complemented tad1-2 line (Compl.). Seven-day-old seedlings raised on synthetic medium were transferred to soil and grown under different environmental conditions in the greenhouse. (A) Phenotype after growth for 15 days under long-day conditions. (B) Phenotype after 21 days under short-day conditions. (C) Twenty-day-old plants grown on soil under short-day conditions were cold stressed at 4°C for 20 days. (D) Sixteen-day-old plants grown on soil under short-day conditions were exposed to heat stress at 30°C for 8 days. (E) Fresh weight of rosettes from plants grown as in (A). (F) Fresh weight of rosettes from plants grown as in (B). (G) Fresh weight of rosettes from plants grown as in (C). (H) Fresh weight of rosettes from plants grown as in (D). Asterisks indicate statistically significant differences (P < 0.05) in Student’s _t_-test. Error bars represent the standard deviation (n = 16 or 17).

Figure 6.

Figure 6.

Measurement of respiration activity and total protein content in tad1 mutant plants, wild-type plants (WT) and the complemented tad1-2 line (Compl.). (A) Leaf respiration rates of 30-day-old plants grown under short-day conditions. (B) Leaf respiration rates of cold stressed plants. Twenty-day-old plants grown in soil under short-day conditions were cold stressed at 4°C for 20 days. Respiration was measured in the dark and is given in nanomoles consumed oxygen per minute and gram fresh weight (FW). Error bars represent the standard deviation (n = 8). Asterisks indicate statistically significant differences (P < 0.05) in Student’s _t_-test. (C) Analysis of total soluble proteins amounts in leaves (n = 6) from cold-stressed plants. (D) Analysis of total soluble proteins amounts in roots (n = 6) from cold-stressed plants.

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