Species-Dependent Regulation of Excitability by RNA Editing

所有现代生物学都是基于遗传信息存储在基因中并在蛋白质中实现的原则。令人惊讶的是，最近的基因组测序项目表明，完全不同的生物体，如人类，苍蝇和蠕虫，携带或多或少共同的基因组。那么，复杂性的遗传基础是什么呢？RNA编辑是一种改变和增加遗传信息的过程，显然可以发挥重要作用，但其生物学意义仍然知之甚少。一种由mRNA中腺苷（A）残基的水解脱氨基介导的编辑形式在所有后生动物的神经系统中普遍存在。通过将A改变为肌苷（I），其被核糖体读取为鸟嘌呤（G），密码子可以突变，蛋白质结构和功能改变。在哺乳动物中，已经确定了相对较少的A-I编辑的mRNA底物，大多数编码参与突触传递的蛋白质。然而，最近的研究已经在果蝇和Loligo中发现了数量惊人的底物，这表明无脊椎动物中的编辑是一个特别强大的过程。这些例子中的许多是离子通道转录物。编辑允许来自单个基因的多个蛋白质。不同的生物如何以及何时进行编辑？哪些mRNA被靶向，蛋白质功能如何改变？就行为而言，头足类动物是最复杂的无脊椎动物。该提案中概述的实验将比较在四种密切相关的鱿鱼物种的巨大轴突中表达的K+通道mRNA是如何编辑的。选择这些物种是因为它们的栖息地跨越了很大的温度梯度，生理研究已经确定它们的钾电导率随温度而变化。早期的研究在乳枪乌贼的K+通道mRNA中发现了14个编辑位点。这些编辑以不同的方式影响通道功能，调节电压依赖性门控，整体K+电导和亚基四聚化。初步证据表明，这些位点的位置和A-I转换频率在物种之间可能会有所不同。在目前的资助期间，实验将使用广泛的方法研究物种特异性编辑的分子基础和功能后果。 分子技术将被用来映射编辑网站和相关的A-I转换频率。生物化学和分子技术将被用来确定关键的二级结构，围绕编辑的腺苷和调节他们的脱氨。生物物理技术，包括宏观和单通道记录，将用于研究编辑位点引起的氨基酸变化如何影响通道功能。总之，这些方法将用于研究物种之间的编辑模式如何变化，这些差异背后的生物化学和分子特性，以及物种特异性编辑如何影响K+通道的生理特性。这些数据很重要，因为它们为A-I编辑如何影响神经功能的进化提供了一个窗口。

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.