Abstract
The mammalian adenosine deaminases acting on RNA (ADARs) constitute a family of sequence-related proteins involved in pre-mRNA editing of nuclear transcripts through site-specific adenosine modification. We report here the identification and characterization of a human ADAR protein, hADAT1, that specifically deaminates adenosine 37 to inosine in eukaryotic tRNA
Ala
. It represents the functional homologue of the recently identified yeast protein Tad1p [Gerber, A., Grosjean, H., Melcher, T. & Keller, W. (1998)
EMBO J.
17, 4780–4789]. The hADAT1 cDNA predicts a protein of 502 aa whose sequence displays strongest overall homology to a
Drosophila melanogaster
ORF (50% similarity, 32% identity), and the catalytic domain is closely related to the other ADAR proteins.
In vitro
, the recombinantly expressed and purified hADAT1 protein efficiently and specifically deaminates A
37
in the anticodon loop of tRNA
Ala
from higher eukaryotes and with lower efficiency from lower eukaryotes. It does not modify adenosines residing in double-stranded RNA or in pre-mRNAs that serve as substrates for ADAR1 or ADAR2. The anticodon stem–loop of tRNA
Ala
alone is not a functional substrate for hADAT1. The enzyme is expressed ubiquitously in human tissues and is represented by a single gene. The identification and cloning of hADAT1 should help to elucidate the physiological significance of this unique modification in tRNA
Ala
, which is conserved from yeast to man.