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Abstract Coleoid cephalopods (octopus, squid and cuttlefish) have unusually complex nervous systems. The coleoid nervous system is also the only one currently known to recode the majority of expressed proteins through A-to-I RNA editing. The deamination of adenosine by adenosine deaminase acting on RNA (ADAR) enzymes produces inosine, which is interpreted as guanosine during translation. If this occurs in an open reading frame, which is the case for tens of thousands of editing sites in coleoids, it can recode the encoded protein. Here, we describe recent findings aimed at deciphering the mechanisms underlying high-level recoding and its adaptive potential. We describe the complement of ADAR enzymes in cephalopods, including a recently discovered novel domain in sqADAR1. We further summarize current evidence supporting an adaptive role of high-level RNA recoding in coleoids, and review recent studies showing that a large proportion of recoding sites is temperature-sensitive. Despite these new findings, the mechanisms governing the high level of RNA recoding in coleoid cephalopods remain poorly understood. Recent advances using genome editing in squid may provide useful tools to further study A-to-I RNA editing in these animals. 

The coleoid cephalopods display unusually extensive mRNA recoding by adenosine deamination, yet the underlying mechanisms are not well understood. Because the adenosine deaminases that act on RNA (ADAR) enzymes catalyze this form of RNA editing, the structure and function of the cephalopod orthologs may provide clues. Recent genome sequencing projects have provided blueprints for the full complement of coleoid cephalopod ADARs. Previous results from our laboratory have shown that squid express an ADAR2 homolog, with two splice variants named sqADAR2a and sqADAR2b and that these messages are extensively edited. Based on octopus and squid genomes, transcriptomes, and cDNA cloning, we discovered that two additional ADAR homologs are expressed in coleoids. The first is orthologous to vertebrate ADAR1. Unlike other ADAR1s, however, it contains a novel N-terminal domain of 641 aa that is predicted to be disordered, contains 67 phosphorylation motifs, and has an amino acid composition that is unusually high in serines and basic amino acids. mRNAs encoding sqADAR1 are themselves extensively edited. A third ADAR-like enzyme, sqADAR/D-like, which is not orthologous to any of the vertebrate isoforms, is also present. Messages encoding sqADAR/D-like are not edited. Studies using recombinant sqADARs suggest that only sqADAR1 and sqADAR2 are active adenosine deaminases , both on perfect duplex dsRNA and on a squid potassium channel mRNA substrate known to be edited in vivo . sqADAR/D-like shows no activity on these substrates. Overall, these results reveal some unique features in sqADARs that may contribute to the high-level RNA recoding observed in cephalopods. 

The coleoid cephalopods have the largest brains, and display the most complex behaviors, of all invertebrates. The molecular and cellular mechanisms that underlie these remarkable advancements remain largely unexplored. Early molecular cloning studies of squid ion channel transcripts uncovered an unusually large number of A→I RNA editing sites that recoded codons. Further cloning of other neural transcripts showed a similar pattern. The advent of deep-sequencing technologies and the associated bioinformatics allowed the mapping of RNA editing events across the entire neural transcriptomes of various cephalopods. The results were remarkable: They contained orders of magnitude more recoding editing sites than any other taxon. Although RNA editing sites are abundant in most multicellular metazoans, they rarely recode. In cephalopods, the majority of neural transcripts are recoded. Recent studies have focused on whether these events are adaptive, as well as other noncanonical aspects of cephalopod RNA editing.