Species-Dependent Regulation of Excitability by RNA Editing

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All modern biology is based on the principle that genetic information is stored in genes and realized in proteins. Surprisingly, recent genome sequencing projects indicate that drastically different organisms, such as humans, flies and worms, carry a more or less common set of genes. What then is the genetic basis of complexity? RNA editing, a process that changes and increases genetic information, could obviously play an important role, however its biological significance remains poorly understood. One form of editing, mediated by the hydrolytic deamination of adenosine (A) residues in mRNAs, is prevalent in the nervous system of all metazoans. By changing A to Inosine (I), which is read by the ribosome as guanine (G), codons can be mutated and protein structure and function changed. In mammals, relatively few mRNA substrates for A-I editing have been identified, most encoding proteins involved in synaptic transmission. More recent investigations, however, have identified a surprisingly large number of substrates in Drosophila and Loligo, suggesting that editing in invertebrates is a particularly robust process. Many of these examples are ion channel transcripts. Editing permits multiple proteins from a single gene. How and when do different organisms edit? Which mRNAs are targeted and how is protein function changed?In terms of behavior, cephalopods are the most sophisticated invertebrates. Experiments outlined in this proposal will compare how K+ channel mRNAs, expressed in the giant axons of four closely related species of squid, are edited. These species were chosen because their habitats span a large thermal gradient, and physiological studies have determined that their potassium conductance varies according to temperature. Earlier investigations identified fourteen editing sites in a K+ channel mRNA from Loligo opalescens. These edits influence channel function in diverse ways, regulating voltage-dependent gating, overall K+ conductance, and subunit tetramerization. Preliminary evidence suggests that the position, and A-I conversion frequency, of some of these sites can vary between species. During the present funding period, experiments will examine the molecular basis, and functional consequences, of species-specific edits, using a broad range of approaches. Molecular techniques will be used to map editing sites and the associated A-I conversion frequency. Biochemical and molecular techniques will be used to identify the critical secondary structure that surrounds edited adenosines and regulates their deamination. Biophysical techniques, including both macroscopic and single channel recordings, will be used to study how the amino acid changes caused by editing sites affect channel function. Taken together, these approaches will be used to investigate how the pattern of editing changes between species, the biochemical and molecular properties underlying these differences, and how species-specific edits affect the physiological properties of K+ channels. These data are important because they provide a window on how A-I editing influences the evolution of nervous function.