Abstract
Simple SummaryAdenosine deaminase acting on RNA (ADAR) is a key enzyme in the editing of adenosine into inosine (A-to-I). The loss or dysfunction of ADAR enzymes in higher eukaryotes affects the editing efficiency of target genes, leading to some neurological diseases. The silkworm Bombyx mori is an oligophagous economically important insect and has been used as an important lepidoptera model insect. By far, the knowledge about A-to-I RNA editing and ADAR members in B. mori (BmADAR) is very limited. In this paper, we present a first molecular comprehensive cloning, sequence analysis of BmADAR transcripts and subcelluar localization of BmADARa. As a result, we obtained six BmADAR transcripts encoding different amino and carboxyl termini, among which BmADARa is a mainly expressed transcript with complete open reading frame. Our further investigations showed that the majority of BmADARa protein exists in the nucleus and has editing function to a specific site of the silkworm synaptotagmin I gene. Overall, by molecular cloning and functional identifing, this paper introduces the first ADAR enzyme in B. mori and contributes to further exploration of the functional domain of BmADARa and its editing substrates and target sites.The most common type of RNA editing in metazoans is the deamination of adenosine into inosine (A-to-I) catalyzed by the adenosine deaminase acting on the RNA (ADAR) family of proteins. The deletion or dysfunction of ADAR enzymes in higher eukaryotes can affect the efficiency of substrate editing and cause neurological disorders. However, the information concerning A-to-I RNA editing and ADAR members in the silkworm, Bombyx mori (BmADAR), is limited. In this study, a first molecular comprehensive cloning and sequence analysis of BmADAR transcripts was presented. A complete open reading frame (ORF) (BmADARa) was obtained using RT-PCR and RACE and its expression pattern, subcellular localization and A-to-I RNA-editing function on the silkworm synaptotagmin I (BmSyt I) were investigated. Subcellular localization analysis observed that BmADARa was mainly localized in the nucleus. To further study the A-to-I RNA-editing function of BmADARa, BmSyt I-pIZ-EGFP was constructed and co-transfected with BmADARa-pIZ-EGFP into BmN cells. The result demonstrates that BmADARa can functionally edit the specific site of BmSyt I. Taken together, this study not only provides insight into the function of the first ADAR enzyme in B. mori, but also lays foundations for further exploration of the functional domain of BmADARa and its editing substrates and target sites.
Highlights
As an RNA-editing enzyme involved in the nucleotide conversion from adenosine to inosine in double-stranded RNA substrates, adenosine deaminase that acts on RNA (ADAR) was first reported and identified in Xenopus laevis [1] and has been cloned and characterized in many metazoans, including mammals, birds, fish, flies and worms [2,3]
A 1900 bp cDNA fragment was amplified by using Reverse transcription PCR (RT-PCR) with primers designed according to the sequences of Bombyx mori adenosine deaminase acting on the RNA (ADAR) (Gene ID: 100101209)
The deamination of adenosine into inosine (A-to-I) is catalyzed by the adenosine deaminase acting on the RNA (ADAR) protein family
Summary
As an RNA-editing enzyme involved in the nucleotide conversion from adenosine to inosine in double-stranded RNA (dsRNA) substrates, adenosine deaminase that acts on RNA (ADAR) was first reported and identified in Xenopus laevis [1] and has been cloned and characterized in many metazoans, including mammals, birds, fish, flies and worms [2,3]. Adenosine to inosine (A-to-I) RNA editing has the capacity to diversify gene expression by the alteration of protein sequences, splicing patterns and base-pairing properties [4,5], which is an important mechanism for the occurrence of protein molecular diversity [6]. In Drosophila, 972 new editing sites in 561 genes were identified, most of which involve neurotransmission [10]
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