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.