An Adenosine Deaminase that Generates Inosine at the Wobble Position of tRNAs

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Science  05 Nov 1999:
Vol. 286, Issue 5442, pp. 1146-1149
DOI: 10.1126/science.286.5442.1146


Several transfer RNAs (tRNAs) contain inosine (I) at the first position of their anticodon (position 34); this modification is thought to enlarge the codon recognition capacity during protein synthesis. The tRNA-specific adenosine deaminase of Saccharomyces cerevisiae that forms I34 in tRNAs is described. The heterodimeric enzyme consists of two sequence-related subunits (Tad2p/ADAT2 and Tad3p/ADAT3), both of which contain cytidine deaminase (CDA) motifs. Each subunit is encoded by an essential gene (TAD2 and TAD3), indicating that I34is an indispensable base modification in elongating tRNAs. These results provide an evolutionary link between the CDA superfamily and RNA-dependent adenosine deaminases (ADARs/ADATs).

It has been known for 35 years that inosine occurs at the wobble position of tRNA anticodons (1, 2), and it was postulated that these tRNAs can translate three codons ending in U, C, or A (3). This important modification occurs in eight cytoplasmic tRNAs in higher eukaryotes (seven in yeast) and in tRNA2 Arg from prokaryotes and plant chloroplasts (4). I34 is thereby formed by hydrolytic deamination of a genomically encoded adenosine (A), and the enzymatic activity for this RNA editing reaction has been partially purified from yeast (5, 6). A family of mammalian adenosine deaminases (ADARs) that convert A to I in double-stranded RNA (dsRNA) and edit diverse cellular mRNA precursors (pre- mRNAs) has been identified (7), but none of these enzymes forms I in tRNA. Recently, a yeast and human protein that acts on tRNA has been cloned by sequence homology to the deaminase domain of the ADAR proteins. This protein, termed Tad1p/ADAT1, specifically deaminates A at position 37 (3′ of the anticodon) in eukaryotic tRNAAla(8, 9). The yeast TAD1 gene is not essential for cell viability, and the function of I37 in tRNA1 Ala, which is further methylated toN 1-methylinosine, is unknown (8).

A search of the Saccharomyces cerevisiae genome for open reading frames (ORFs) encoding putative deaminases revealed a hypothetical ORF (YJL035c/YJD5) that contains a PROSITE pattern (accession number PS00903) (10) characteristic of cytidine/deoxycytidylate deaminases (CDAs). To investigate the physiological relevance of YJL035c, we deleted one of the two copies of the gene in a diploid yeast strain (11). Sporulation and tetrad analysis of heterozygotes showed that these tad2Δ segregants were not viable.

To confirm that TAD2 is required for vegetative growth, we transformed tad2Δ heterozygotes with pFL38-Tad2 (a centromeric plasmid containing both TAD2 andURA3) or pGAL-FLIS6-Tad2 (a centromeric plasmid with URA3 bearing the coding sequence of TAD2fused to a 5′-FLAG and a 3′-hexahistidine epitope) (12). In both cases dissection of transformants resulted in four viable spores, and tad2Δ segregants containing pFL38-Tad2 or pGAL-FLIS6-Tad2, respectively, were isolated. When these cells were grown on 5-fluoro-orotic acid (5-FOA), which is toxic to cells expressing URA3, no colonies appeared at either 23° or 30°C, showing that TAD2 is an essential gene.

We next isolated a temperature-sensitive (ts) tad2allele to functionally characterize TAD2 (13). Cells bearing the tad2-1 allele grew normally at 23°C but could not form colonies at 37°C. A cell-free extract prepared from these cells lacked specific tRNA:A34 deaminase activity as measured by incubation with synthetic [33P]ATP (adenosine 5′-triphosphate)–labeled yeast tRNA1 Ala (5,8), which is a natural substrate (1,2). Activity was restored by addition of purified recombinant Tad2 protein (rTad2p) to the extract (14). Furthermore, tRNAAla isolated fromtad2-1 mutant cells contained unmodifed A34, indicating that Tad2p is involved in deamination in vivo (14). However, rTad2p alone had no deaminase activity. These results suggested that Tad2p is necessary but not sufficient to form I34 in tRNAs and that an additional component is required.

To isolate this factor, we cultured tad2Δ [pGal-FLIS6-Tad2] cells and purified the Tad2 fusion protein (15). A protein of 38 kD copurified in a 1:1 ratio with the tagged Tad2p (Fig. 1, A and B), and fractions containing both Tad2p and p38 converted A34 to I34 in synthetic tRNAAla (Fig. 1C). To identify p38, the masses of lysine C–digested peptides were determined by mass spectrometry. Nine protein fragments matched the putative S. cerevisiae ORF YLR136c [Munich Information Center for Protein Sequences (MIPS) accession number S53395]. Polymerase chain reaction (PCR) and sequence analysis on yeast cDNA with primers annealing upstream of YLR136c and at the stop codon confirmed the presence of two introns (16). Thus, this gene, named TAD3, represents the fifth gene in S. cerevisiae that contains two introns (17).

Figure 1

Purification of the tRNA A:34 deaminase. (A) Samples (5 μl) of the final MonoQ column fractions were subjected to electrophoresis on a 12% SDS-polyacrylamide gel, and proteins were stained with silver. Load (L) and fraction numbers are indicated at the top, and molecular masses on the left (in kilodaltons). (B) Immunoblot analysis with a mouse anti-FLAG M2 monoclonal antibody (1:5000 dilution). (C) tRNA-specific adenosine deaminase assay (8). [α-33P]ATP-labeled yeast tRNAAla (200 fmol) was incubated with samples (0.2 μl) of column fractions for 45 min at 30°C. After ethanol precipitation the RNA was digested with P1 nuclease and reaction products were separated by one-dimensional thin-layer chromatography. The chromatographic origin and the migration positions of adenosine monophosphate (AMP) and inosine monophosphate (IMP) are indicated on the left.

We tested the functionality of these cDNAs in vivo by disrupting one allele of TAD3 (11). Tetrad dissection of the heterozygotes showed that tad3Δ segregants were not viable. tad3Δ segregants containing the plasmid pFL38-Tad3 (CEN-URA3) (16) could be isolated. After plating the cells on 5-FOA, the cells grew only whenTAD3 was provided on a second plasmid with LEU2(pFL36-Tad3) or ADE2 (pGal-FL-Tad3) markers (18). In contrast, tad3Δ cells that expressed Tad3p from the intronic ATG2 (ΔNTad3p) had a slight growth defect at 23°C and were nonviable at 37°C (18). Extracts from these ts cells (tad3-1) lacked tRNA:A34 editing activity, indicating that Tad3p is essential for A34 deamination (14). The requirement of TAD2 andTAD3 for cell viability strongly suggests that inosine at the wobble position of tRNAs is an essential modification in yeast.

To confirm that Tad2p and Tad3p compose the tRNA:A34 deaminase, we reconstituted the activity with purified recombinant proteins (Fig. 2) (19). The combination of rTad2p and rTad3p, but neither protein alone, specifically converted A34 to I34 in tRNAs from yeast and the silkwormBombyx mori (Fig. 2, B and D). In addition, the in vitro–reconstituted deaminase was active on Escherichia coli tRNA2 Arg, yeast tRNA2 Ser(another natural substrate), and a mutant yeast tRNAAsp in which the anticodon loop was exchanged with that of yeast tRNA2 Arg (5). In contrast, removal of one base from the 7-nucleotide anticodon loop in yeast tRNAAla(ΔU33) abolished deaminase activity (14). A mixture of Tad2p and Tad3p, but neither protein alone, could be ultraviolet (UV) cross-linked to yeast tRNAAla (Fig. 2E) orB. mori tRNAAla (14). This may indicate that association of the two subunits is required for tRNA binding. To further analyze the Tad2p-Tad3p complex, we combined the recombinant proteins and ran them on a sizing column. rTad2p-rTad3p coeluted at an estimated molecular mass of 70 kD together with tRNA:A34 deaminase activity (2), indicating that Tad2p and Tad3p form a heterodimeric complex. This type of subunit composition has not been observed in other deaminases. CDAs form homodimeric or homotetrameric protein complexes (20), whereas ADARs and Tad1p/ADAT1 act as monomers (7, 8).

Figure 2

Reconstitution of the tRNA A:34 deaminase with recombinant Tad2p and Tad3p. (A) Purified rTad2p and rTad3p were separated by SDS-PAGE and stained with Coomassie blue. (B) Recombinant proteins (10 ng) were used in tRNA-specific adenosine deaminase assays (8). Abbreviations: y, yeast; B.m., B. mori; E.c., E. coli; A34G, mutant tRNA containing G34 instead of A34; A37G, G37 instead of A37. (C) Mutant rTad2 and rTad3 proteins were assayed on WT tRNAAla of B. mori. (D) Sequence analysis of edited yeast and E. coli tRNAs. After incubation of synthetic tRNAs with rTad2p-rTad3p or buffer (control), tRNAs were reverse transcribed and amplified by PCR and products were sequenced. Only the anticodon loop-region is shown. Nucleotide position 34 is underlined, and A peaks are dashed. Because inosine base-pairs with C, I is represented as G. (E) UV cross-linking of Tad1p and rTad2p-rTad3p to labeled WT yeast tRNAAla. Reactions (10 μl) were carried out with 200 ng of each recombinant protein and 250 fmol of labeled tRNAAla in assay buffer. After incubation for 15 min at room temperature, the reactions were irradiated on ice in a UV Stratalinker at 400 mJ and digested with 250 ng of ribonuclease A for 30 min at 37°C. Proteins were separated on denaturing SDS–12% polyacrylamide gels and exposed on a PhosphorImager screen (Molecular Dynamics).

The COOH-terminal part of Tad3p is related in sequence to Tad2p (26% amino acid identity and 45% similarity in 120–amino acid overlap) (2). The homologous region contains the deaminase motif signatures found in the CDA superfamily including the catalytic subunit APOBEC-1, which converts C to U in mammalian apolipoprotein B pre-mRNA (20, 21) (Fig. 3). In particular, Tad2p-Tad3p share the three key amino acids (His, Cys, Cys) involved in zinc coordination and the proline in the deaminase motif II that acts as the ammonium group binding site. Furthermore, Tad2p contains a conserved glutamate (Glu56) that is required for proton shuttling during catalysis in CDAs (20). A Tad2p point mutant, where Glu56 was substituted by Ala (E56A) (22), combined with wild-type (WT) Tad3p, had no deaminase activity in vitro (Fig. 2C). In Tad3p, this Glu is replaced by valine (Val218) (framed in Fig. 3B). The Tad3 point mutant V218E acted like the WT protein and could not substitute for the E56A mutation in Tad2p (Fig. 2C). Therefore, Tad2p most likely represents the catalytic subunit of the tRNA:A34 deaminase.

Figure 3

Tad2p and Tad3p contain a deaminase domain that is similar to those of CDAs and ADARs/ADAT1. (A) Protein domain organization of Tad2p and Tad3p. The deaminase domain (DM) is boxed in gray. A putative nuclear localization signal (NLS) in Tad3p (black box), the region of sequence similarity between Tad2p and Tad3p (dashed line), and the lengths of the proteins (in amino acids) are indicated. (B) Multiple sequence alignment of deaminase domains. Highly conserved residues found in at least two different enzymes are framed in black; similar ones are in gray. The deaminase motifs I, II, and III are overlined, and the putative Zn2+-chelating residues (#) and proton-transferring amino acid (o) are indicated. The DNA Data Bank of Japan/European Molecular Biology Laboratory/GenBank accession numbers are indicated in the column next to the names of the enzymes. Abbreviations:C. elegans, Caenorhabditis elegans;E. coli, Escherichia coli;H. infl., Haemophilus influenzae;B. sub., Bacillus subtilis;P. ging., Porphyromonas gingivalis;A. aeolicus, Aquifax aeolicus; andR. prowazekii, Rickettsia prowazekii. Abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.

Database searches identified a number of sequences related to Tad2p and Tad3p, suggesting that these putative deaminases are orthologs (Fig. 3) (2). Remarkably, the prokaryotic genomes encoded only one such homologous polypeptide. Therefore, Tad2p and Tad3p may be paralogs that appeared after the divergence of prokaryotes and eukaryotes by genome duplication. Through this event and further genetic drift, the eukaryotic tRNA:A34 deaminases could have acquired the ability to modify additional tRNA substrates. This is consistent with the fact that the E. coli tRNA:A34 deaminase cannot modify any of the seven yeast tRNAs containing I34, whereas the yeast enzyme can modify E. coli tRNA2 Arg(5).

The Tad2-Tad3 protein family appears to be positioned evolutionarily between the proteins of the CDA superfamily and the members of the ADAR family including Tad1p/ADAT1. Tad2p-Tad3p share the indicative deaminase motifs of CDAs and form a heterodimeric complex. But in contrast to CDAs, Tad2p and Tad3p do not deaminate free cytidine or cytosine in vitro (23, 14). Moreover, Tad2p-Tad3p functionally belong to the ADAR family. ADARs share the deaminase motif I and II of CDAs but have a conserved deaminase motif III that contains the third putative Zn2+-chelating residue required for catalysis (24) (Fig. 3).

On the basis of this sequence relationship between CDAs and ADARs and considering the structural differences between CDA (21) and ADA (25), it was hypothesized that ADARs evolved from a CDA precursor (26). Our results substantiate this hypothesis and we further propose that Tad2p-Tad3p is the ancestor of the deaminase domain found in ADARs and Tad1p. After the divergence of prokaryotes and eukaryotes, a Tad2p-like enzyme might have further evolved by establishment of deaminase motif III and changing site specificity from position 34 to position 37 in one tRNA substrate. This Tad1p-like protein might then have evolved to the metazoan ADARs by the acquisition of dsRNA-binding modules (8).

  • * To whom correspondence should be addressed. E-mail: walter.keller{at}


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