title: A molecular, phylogenetic and functional study of thedADARmRNA truncated isoform duringDrosophilaembryonic development reveals an editing-independent function.
abstract: Adenosine Deaminases Acting on RNA (ADARs) have been studied in many animal phyla, where they have been shown to deaminate specific adenosines into inosines in duplex mRNA regions. InDrosophila, two isoform classes are encoded, designated full-length (contains the editase domain) and truncated (lacks this domain). Much is known about the full-length isoform, which plays a major role in regulating functions of voltage-gated ion channel proteins in the adult brain. In contrast, almost nothing is known about the functional significance of the truncated isoform.In situhybridization shows that both isoform mRNA classes are maternally derived and transcripts for both localize primarily to the developing central nervous system. Quantitative RT-PCR shows that about 35% of alldADARmRNA transcripts belong to the truncated class in embryos. 3'-RACE results show that abundance of the truncated isoform class is developmentally regulated, with a longer transcript appearing after the mid-blastula transition. 3'-UTR sequences for the truncated isoform have been determined from diverseDrosophilaspecies and important regulatory regions including stop codons have been mapped. Western analysis shows that both mRNA isoform classes are translated into protein during embryonic development, as full-length variant levels gradually diminish. The truncated protein isoform is present in everyDrosophilaspecies studied, extending over a period spanning about 40 × 106years, implying a conserved function. Previous work has shown that adADARprotein isoform binds to the evolutionarily conservedrnp-4fpre-mRNA stem-loop located in the 5'-UTR to regulate splicing, while no RNA editing was observed, suggesting the hypothesis that it is the non-catalytic truncated isoform which regulates splicing. To test this hypothesis, we have utilized RNAi technology, the results of which support the hypothesis. These results demonstrate a novel, non-catalytic function for the truncateddADARprotein isoform inDrosophilaembryonic development, which is very likely evolutionarily conserved.
authors.affiliation: Department of Biology, Cell Molecular and Structural Biology Program, Miami University, Oxford, USA.
authors.forename: Sushmita
authors.initials: S
authors.lastname: Ghosh
authors.affiliation: Department of Biology, Cell Molecular and Structural Biology Program, Miami University, Oxford, USA.
authors.forename: Yaqi
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Biology, Cell Molecular and Structural Biology Program, Miami University, Oxford, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Cook
authors.affiliation: Department of Biology, Cell Molecular and Structural Biology Program, Miami University, Oxford, USA.
authors.forename: Lea
authors.initials: L
authors.lastname: Chhiba
authors.affiliation: Department of Biology, Cell Molecular and Structural Biology Program, Miami University, Oxford, USA.
authors.forename: Jack C
authors.initials: JC
authors.lastname: Vaughn
keywords: 5′-UTR Intron Retention; RNAi Knockdown; Truncated dADAR Isoform; dADAR Gene; rnp-4f Gene