An extra double-stranded RNA binding domain confers high activity to a squid RNA editing enzyme.
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.
Palavicini
Juan Pablo
JP
Institute of Neurobiology, University of Puerto Rico Medical Sciences Campus, San Juan, Puerto Rico 00901.
O'Connell
Mary A
MA
Rosenthal
Joshua J C
JJ
D000243:Adenosine Deaminase / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D049832:Decapodiformes; D008969:Molecular Sequence Data; D017393:RNA Editing; D012330:RNA, Double-Stranded / Q000737:chemistry* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D016415:Sequence Alignment
D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C567955:ADARB1 protein, human; D000243:Adenosine Deaminase