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. 2007 May 8;104(19):7821-6.
doi: 10.1073/pnas.0702394104. Epub 2007 May 1.

An adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U deamination of DNA

Mary Anne T Rubio  1 , Irena Pastar, Kirk W Gaston, Frank L Ragone, Christian J Janzen, George A M Cross, F Nina Papavasiliou, Juan D Alfonzo
Affiliations

An adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U deamination of DNA

Mary Anne T Rubio et al. Proc Natl Acad Sci U S A. .

Abstract

Adenosine-to-inosine editing in the anticodon of tRNAs is essential for viability. Enzymes mediating tRNA adenosine deamination in bacteria and yeast contain cytidine deaminase-conserved motifs, suggesting an evolutionary link between the two reactions. In trypanosomatids, tRNAs undergo both cytidine-to-uridine and adenosine-to-inosine editing, but the relationship between the two reactions is unclear. Here we show that down-regulation of the Trypanosoma brucei tRNA-editing enzyme by RNAi leads to a reduction in both C-to-U and A-to-I editing of tRNA in vivo. Surprisingly, in vitro, this enzyme can mediate A-to-I editing of tRNA and C-to-U deamination of ssDNA but not both in either substrate. The ability to use both DNA and RNA provides a model for a multispecificity editing enzyme. Notably, the ability of a single enzyme to perform two different deamination reactions also suggests that this enzyme still maintains specificities that would have been found in the ancestor deaminase, providing a first line of evidence for the evolution of editing deaminases.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Schematic representation of active-site core domains of the T. brucei tRNA-editing deaminase. The T. brucei sequences are compared with representative members of the cytidine and adenosine deaminase family. Although TbADATA2p and ADAT3p are required for adenosine deamination in tRNA, their active site resembles that of CDA. The figure shows the conserved residues shared by RNA editing adenosine and cytidine deaminases compared with tbtad2p and tbtad3p (boldface letters). Boxes, putative active sites of TbADAT2p and TbADAT3p. Asterisks, conserved residues involved in Zn2+ coordination found in all nucleotide- and RNA-editing deaminases. Arrows, absolutely conserved glutamate residue that is involved in proton shuttling during deamination; this residue is absent in tbtad3p, demonstrating that, as in yeast, tbtad3p may only serve a structural role in inosine formation. Vertical lines, shared conserved residues found in tbtad2p and CDAs. Diagonal line, altered position of one of the essential Zn2+ coordinating domains typical of RNA-editing adenosine deaminases.
Fig. 2.
Fig. 2.
Induction of RNAi against TbADAT2 causes a growth phenotype and reduces the levels of TbADAT2 within cells. (A) Effect of RNAi induction by tetracycline on T. brucei growth curve. (B) Western blot analysis with anti-TbADAT2 antibodies of whole cell extract. Extracts from +Tet cells were prepared 24 and 48 h after tetracycline addition to the growth medium. Anti-tubulin antibodies were used as control for protein levels. (C) Total RNA was isolated from the −Tet and +Tet (48 h after induction) cells for RT-PCR with tRNAThr(AGU)-specific primers. M, DNA size marker for electrophoresis. (D) RT-PCR products from C were purified and cloned into a plasmid vector, and independent clones were sequenced to assess editing levels.
Fig. 3.
Fig. 3.
TbADAT2 is required for A-to-I editing of tRNAThr in T. brucei. (A) Whole-cell extracts from uninduced and induced [−Tet vs. +Tet (48h)] cells tested for in vitro A-to-I editing activity as described previously (11). tRNAThr(AGU) radiolabeled at every adenosine was incubated with protein fractions for increasing times. Reaction products were purified, digested with nuclease P1, and separated by TLC (11). Black circles, positions of 5′ inosine monophosphate (pI) and 5′ adenosine monophosphate (pA) markers for TLC (visualized by UV shadowing). Arrow, expected position for pI. Bracket, position of extra spots observed with the RNAi-induced extract; their significance is unknown. (B) Plot of the percent conversion at adenosine-34 per μg of protein vs. time from the +Tet (▵) and −Tet (48 h) (◇) cells. Specific conversion at A34 is calculated by normalizing the amount of radioactivity at A34 to the total number of label adenosines (n = 14), yielding a maximum theoretical value of 7.1% (11). (C) TLC analysis as in A but with recombinant TbADAT2/3 coexpressed in E. coli and purified through affinity- and size-exclusion chromatography. Lane 1, radioactive tRNAThr alone; lane 2, tRNA incubated with TbADAT2/3; lane 3, tRNAThr (A34 replaced by G34; a specificity control) incubated with TbADAT2/3; lane 4, tRNAThr incubated with recombinant EcADATa; lane 5, tRNAArg from E. coli (EctRNAArg) alone; lane 6, EctRNAArg incubated with recombinant EcADATa; lane 7, EctRNAArg (G34 specificity control) incubated with EcADATa; lane 8, EctRNAArg containing G34, alone; lane 9, tRNAThr(AGU) incubated with the recombinant TbADAT2(C136A)/ADAT3 mutant complex coexpressed in E. coli. (D) Two-dimensional TLC analysis of the reactions in lanes 1 and 2 from C to corroborate the identity of the pA and pI spots. Two different solvent systems were used as described (11). −E and +E refer to tRNAs incubated in the presence or absence of TbADAT2/3 enzyme as in C. The arrows and A and B refer to the different buffers system used (11). (Right) Expected migration of the different nucleotides according to published maps. Broken circles, positions of the different cold markers visualized by UV shadowing. pA, pC, pG, pU, and pI, 5′ nucleotide monophosphates for adenosine, cytosine, guanosine, and uridine, respectively.
Fig. 4.
Fig. 4.
TbADAT2 mutates deoxycytidine in E. coli and mediates C-to-U deamination in ssDNA. (A) Mutation frequencies for the rpoB gene in E. coli BH156 transformed with TbADAT2, hAID (a positive control), TbADAT2 (C136A, an active-site mutant), EcADATa (a bacterial ADAT), TbADAT3 alone, or empty vector. Each mark represents the frequency of mutation of an independent culture induced overnight with anhydrotetracycline (AHT); mean values are given for each expression vector. Mutation frequencies were calculated by scoring for the appearance of RifR colonies vs. total transformants. (B) Recombinant TbADAT2/3 or the TbADAT2(C136A)/ADAT3 complex was incubated with ssDNA, with every cytosine radioactively labeled. (Left) One-hour incubation. (Right) Time course for the indicated times. BSA, negative control where the ssDNA substrate was incubated with BSA. pC, pU, positions of cold 5′ cytosine monophosphate and 5′ uridine monophosphate markers visualized by UV. (C) Distribution of independent RifR mutations found in cells transformed with a TbADAT2 expression construct. The rpoB sequence from aa 507–539 is shown. Shaded boxes, preferred sites in AID-expressing cells; deaminated nucleotides are underlined. Lowercase letters, type of nucleotide change observed in each mutant. The nature of the Rif mutants was determined by directly amplifying and sequencing the relevant section of rpoB as described (19).
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