RESUMO
The specificity and extent of RNA editing by ADAR enzymes is determined largely by local primary sequence and secondary structural imperfections in duplex RNA. Here we surgically alter conserved cis elements associated with a cluster of ADAR modification sites within the endogenous Drosophila paralytic transcript. In addition to the local requirement for a central imperfect RNA duplex containing the modified adenosines, we demonstrate that a secondary RNA duplex containing splicing signals strongly modulates RNA editing. A subtle non-coding mutation, extending base pairing of this accessory helix, confers significant phenotypic consequences via effects on splicing. Through mutation/counter-mutation, we also uncover and functionally replace a highly conserved intronic long-range tertiary pseudoknot that is absolutely required for deamination of one particular adenosine in the central duplex. Our results demonstrate that complex RNA tertiary structures, which may be difficult to predict computationally, form in vivo and can regulate RNA-editing events.
Assuntos
Conformação de Ácido Nucleico , Edição de RNA/genética , Adenosina Desaminase/metabolismo , Alelos , Animais , Sequência de Bases , Sequência Conservada/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Éxons/genética , Feminino , Genótipo , Íntrons/genética , Masculino , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Splicing de RNA/genética , Canais de Sódio/metabolismoRESUMO
Discovering RNA editing sites in model organisms provides an insight into their adaptations in addition to finding potential sites for the regulation of neural activity and the basis of integrated models of metazoan editing with a variety of applications, including potential clinical treatments of neural dysregulation. The zebrafish, Danio rerio, is an important vertebrate model system. We focused on the grin1b gene of zebrafish due to its important function in the nervous tissue as a glutamate receptor. Using a comparative sequence-based approach, we located possible RNA editing events within the grin1b transcript. Surprisingly, sequence analysis also revealed a new editing site which was not predicted by the comparative approach. We here report the discovery of two novel RNA editing events in grin1b transcripts of embryonic zebrafish. The frequency of these editing events and their locations within the grin1b transcript are also described.
Assuntos
Proteínas do Tecido Nervoso/genética , Edição de RNA , Receptores de N-Metil-D-Aspartato/genética , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Transcrição Gênica , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Adenosine-to-Inosine RNA editing introduces changes in RNA transcripts via a post-transcriptional mechanism, the hydrolytic deamination of adenosine (A) to inosine (I) which is interpreted as guanosine by cellular machineries. Adenosine deaminases that act on RNA (ADAR) enzymes catalyze editing in double-stranded (ds) RNA substrates.
Assuntos
Adenosina/genética , Inosina/genética , Edição de RNA/genética , Adenosina/química , Animais , Variação Genética/genética , Genoma/genética , Humanos , Inosina/químicaRESUMO
An unknown number of precursor messenger RNAs undergo genetic recoding by modification of adenosine to inosine, a reaction catalyzed by the adenosine deaminases acting on RNA (ADARs). Discovery of these edited transcripts has always been serendipitous. Using comparative genomics, we identified a phylogenetic signature of RNA editing. We report the identification and experimental verification of 16 previously unknown ADAR target genes in the fruit fly Drosophila and one in humans-more than the sum total previously reported. All of these genes are involved in rapid electrical and chemical neurotransmission, and many of the edited sites recode conserved and functionally important amino acids. These results point to a pivotal role for RNA editing in nervous system function.
Assuntos
Adenosina Desaminase/metabolismo , Drosophila/genética , Genes de Insetos , Canais Iônicos/genética , Edição de RNA , Transmissão Sináptica , Adenosina/metabolismo , Animais , Sequência de Bases , Drosophila melanogaster/genética , Genômica , Humanos , Inosina/metabolismo , Ativação do Canal Iônico , Canais Iônicos/metabolismo , Dados de Sequência Molecular , Sistema Nervoso/metabolismo , Filogenia , Canais de Potássio/genética , Canais de Potássio/metabolismo , Proteínas de Ligação a RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SinapsesRESUMO
A comparison of the activity of genetic elements from the regulatory region of the Drosophila melanogaster Deformed gene during embryogenesis and adult life reveals important similarities and differences. The 2.7 kb epidermal autoregulatory enhancer (EAE) of the Deformed gene drives expression of a beta-galactosidase reporter in unique spatial and temporal patterns in the adult antennae; this pattern is insensitive to temperature effects. The Deformed regulatory region possesses distinct enhancer elements that can direct the expression of a beta-galactosidase reporter spatially and temporally. A 120 bp region can reproduce the general features of the larger EAE fragment. The Deformed binding site is essential for temporal and spatial expression of beta-galactosidase during embryogenesis but is not required in the adult.
