The Properties of a tRNA-Specific Adenosine Deaminase from Drosophila melanogaster Support an Evolutionary Link between Pre-mRNA Editing and tRNA Modification

Abstract

Pre-mRNA editing involving the conversion of adenosine to inosine is mediated by adenosine deaminases that act on RNA (ADAR1 and ADAR2). ADARs contain multiple double-stranded RNA(dsRNA)-binding domains in addition to an adenosine deaminase domain. An adenosine deaminase acting on tRNAs, scTad1p (also known as scADAT1), cloned from Saccharomyces cerevisiae has a deaminase domain related to the ADARs but lacks dsRNA-binding domains. We have identified a gene homologous to scADAT1 in the region of Drosophila melanogaster Adh chromosome II. Recombinant Drosophila ADAT1 (dADAT1) has been expressed in the yeast Pichia pastoris and purified. The enzyme has no activity on dsRNA substrates but is a tRNA deaminase with specificity for adenosine 37 of insect alanine tRNA. dADAT1 shows greater similarity to vertebrate ADARs than to yeast Tad1p, supporting the hypothesis of a common evolutionary origin for ADARs and ADATs. dAdat1 transcripts are maternally supplied in the egg. Zygotic expression is widespread initially and later concentrates in the central nervous system. Adenosine and cytosine have exocyclic amino groups that participate in Watson-Crick base pairing during transcription and translation. RNA editing enzymes have been discovered that deaminate specific adenosine residues to inosine (4, 32, 38, 40) or specific cytosine residues to uridine in RNA molecules (11,39). This can result in the incorporation of different amino acids at edited positions or the formation of a smaller protein due to the generation of a stop codon. Two closely related adenosine deaminases acting on RNA (ADARs) have been identified in vertebrates (3) that catalyze the deamination of specific adenosine residues to inosine in pre-mRNAs (for a review, see reference 22). These enzymes have homologous adenosine deaminase domains (32) and also contain multiple double-stranded RNA(dsRNA)-binding domains (47). ADARs recognize and deaminate specific adenosines within exons that form duplexes with flanking intronic sequences in pre-mRNA (20, 32). ADAR activity is ubiquitous and has been found in all metazoans tested and in most tissues (52). The abundance of inosine in polyA ϩ RNA has been estimated to be 1 in 17,000 nucleotides in brain and less in other mammalian tissues, correlating with ADAR expression levels (35). Inosine in edited transcripts directs the incorporation of cytosine during firststrand synthesis of cDNA (5), and RNA editing events have usually been identified in cDNA sequences in which guanosine replaces a genomically encoded adenosine (8, 45). Pre-mRNAs encoding subunits of the glutamate-gated ion channels (for a review, see reference 42) and the G protein-coupled serotonin 2C receptor (8) undergo RNA editing of their sequences by this mechanism. Proteins encoded by edited mRNAs often have functional properties that differ from the genomically encoded versions.

RNA Editing by Adenosine Deaminases That Act on RNA

“…The A37 deamination only occurs in eukaryotes, and only on A37 of tRNA ala . A single polypeptide, Tad1p, catalyzes this reaction, and this enzyme has been characterized in yeast (40), humans (41), and flies (42). The Tad2p/Tad3p proteins show sequence similarities to the ADARs, as well as the cytidine deaminases, and as described later, provide strong support for the idea that ADARs evolved from the cytidine deaminase superfamily.…”

Integrated tRNA, transcript, and protein profiles in response to steroid hormone signaling

“…Queuosine modification depends on nutrient availability and leads to increased accuracy of codon recognition, suggesting that environmental context can dramatically influence tRNA modification and consequently translation. Though a tRNA-specific adenosine deaminase, ADAT1, has been identified in D. melanogaster (Keegan et al 2000), the effects of A to I tRNA editing in Drosophila have remained largely uncharacterized.…”

Aminohydrolases Acting on Adenine, Adenosine and Their Derivatives

“…Substrates of diff erent adenosine deaminase enzymes are adenosine 24 and adenine 25 and their derivatives, such as 2',3'-deoxyadenosine 26 33 , tRNA 34 , and other purine compounds as 2-amino-6-chloropurine riboside 28 , 6-chloropurine riboside and 6-methoxypurinriboside 35 , 6-methylaminopurine ribonucleoside 25 , and formycin A 27 . Metals ions in specifi c concentrations are in some cases essential for the activity.…”