An extra double-stranded RNA binding domain confers high activity to a squid RNA editing enzyme
Year: 2009
Type: article
Abstract: RNA editing by adenosine deamination is particularly prevalent in the squid nervous system. We hypothesized that the squid editing enzyme might contain structural differences that help explain this phenomenon. As a first step, a squid adenosine deaminase that acts on RNA (sqADAR2a) cDNA and the gene that encodes it were cloned from the giant axon system. PCR and RNase protection assays showed that a splice variant of this clone (sqADAR2b) was also expressed in this tissue. Both versions are homologous to the vertebrate ADAR2 family. sqADAR2b encodes a conventional ADAR2 family member with an evolutionarily conserved deaminase domain and two double-stranded RNA binding domains (dsRBD). sqADAR2a differs from sqADAR2b by containing an optional exon that encodes an "extra" dsRBD. Both splice variants are expressed at comparable levels and are extensively edited, each in a unique pattern. Recombinant sqADAR2a and sqADAR2b, produced in Pichia pastoris, are both active on duplex RNA. Using a standard 48-h protein induction, both sqADAR2a and sqADAR2b exhibit promiscuous self-editing; however, this activity is particularly robust for sqADAR2a. By decreasing the induction time to 16 h, self-editing was mostly eliminated. We next tested the ability of sqADAR2a and sqADAR2b to edit two K+ channel mRNAs in vitro. Both substrates are known to be edited in squid. For each mRNA, sqADAR2a edited many more sites than sqADAR2b. These data suggest that the "extra" dsRBD confers high activity on sqADAR2a.
Source: RNA
DOI: 10.1261/rna.1471209
Language: English
Topic: RNA regulation and disease
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: bronze
Funders: National Science Foundation, National Institutes of Health, Medical Research Council, National Institute of General Medical Sciences, National Institute of Neurological Disorders and Stroke, National Center for Research Resources, National Institute on Minority Health and Health Disparities, Core Research for Evolutional Science and Technology
Awards: Advancing Competitive Biomedical Research in Puerto Rico, Specialized Program in Cellular & Molecular Neurobiology, G4877463793, Support for Enhancement Excellence in Research at UPR RP, Species-Dependent Regulation of Excitability by RNA Editing, Center for Collaborative Research in Health Disparities, CREST - Center for Tropical Applied Ecology and Conservation at the University of Puerto Rico-Rio Piedras, G8198795195, Center for Collaborative Research in Health Disparities, G9000162437
OpenAlex ID: W2170381624