Regulation of RNA Editing in the CNS

描述(申请人提供)：通过核糖核酸编辑将腺苷转化为肌苷(A-to-L)代表了一种日益公认的转录后机制，用于在真核基因表达中产生多样性。这种RNA修饰已经被证明改变了电压门控和配基门控离子通道的离子渗透、电生理和动力学性质，并调节了受体G-蛋白质相互作用的效果。我们实验室最近的研究表明，ADAR2蛋白的表达受到一种负性自我调节策略的调控，这种策略是由于ADAR2蛋白能够编辑自己的Pre-RNA，从而指导不同的剪接模式。ADAR2蛋白是一种双链RNA特异性腺苷脱氨酶，参与哺乳动物RNA转录本的编辑。基于这些发现，我们开发了两个独立的小鼠模型系统，以进一步了解ADAR2调节在中枢神经系统功能中可能发挥的作用(S)。这些研究的长期目标是确定核糖核酸编辑事件调节中枢神经系统功能的细胞过程，并确定这种A-to-L修饰导致的功能后果。1)错误表达ADAR2转基因的突变小鼠表现出高吞噬介导的成熟期肥胖，并伴有高血糖和高皮质醇血症。为了进一步研究这些表型变化的基础，我们将检查所有经过验证的小鼠ADAR底物的编辑模式的变化，表征已知喂养途径中的表达谱变化，并通过产生仅在神经元或外周靶点过表达ADAR2的小鼠来确定ADAR2在总体能量平衡途径中的位置。2)为了进一步研究ADAR2失调的分子后果，我们还开发了突变小鼠，在这些突变小鼠中，ADAR2编辑自己的Pre-mRNA的能力已经被选择性地消融。将评估突变小鼠不同脑区ADAR2 mRNA和蛋白质水平的变化，以及先前识别的和新的ADAR底物编辑模式的变化。还将评估突变动物对红藻氨酸诱导的癫痫发作和兴奋性毒性损伤的易感性的变化，以及中枢喂养行为的变化。3)最近发现的人类RNA 3‘-非翻译区(3’-UTR)Alu重复元件内的编辑事件的功能后果将与RNA稳定性和核保留的变化有关，以此作为调节蛋白质表达的机制。预计这些研究不仅将有助于确定mRNAs非编码区A-to-L修饰的功能后果，还将为调控ADAR2表达的细胞过程的生理相关性以及它们与参与癫痫易感性、能量平衡和中枢神经系统其他功能的神经递质受体功能的关系提供新的见解。

DESCRIPTION (provided by applicant): The conversion of adenosine to inosine (A-to-l) by RNA editing represents an increasingly recognized post-transcriptional mechanism for generating diversity in eukaryotic gene expression. Such RNA modifications have been shown to alter the ion permeation, electrophysiologic and kinetic properties of both voltage- and ligand-gated ion channels and to modulate the efficacy of receptorG-protein interactions. Recent studies in our laboratory have demonstrated that the expression of ADAR2 protein, a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNA transcripts, is modulated by a negative autoregulatory strategy that results from the ability of this enzyme to edit its own pre-mRNA, thereby directing alternative splicing patterns. Based upon these findings, we have developed two independent mouse model systems to further understand the role(s) that ADAR2 regulation may play in the function of the central nervous system. The long-term objectives of these studies are to identify the cellular processes by which RNA editing events can modulate central nervous system function and to identify the functional consequences resulting from such A-to-l modifications. 1) Mutant mice misexpressing an ADAR2 transgene demonstrate a hyperphagia-mediated, maturity-onset obesity accompanied by hyperglycemia and hypercortisolism. To further examine the bases of these phenotypic alterations, we will examine alterations in editing patterns for all validated murine ADAR substrates, characterize expression profile changes in known feeding pathways and determine where ADAR2 fits into the overall energy balance pathway by generating mice that overexpress ADAR2 solely in neurons or peripheral targets. 2) To further examine the molecular consequences of ADAR2 dysregulation, we have also developed mutant mice in which the ability of ADAR2 to edit its own pre-mRNA has been selectively ablated. Mutant mice will be assessed for changes in ADAR2 mRNA and protein levels in discrete brain regions and for alterations in editing patterns for previously identified and novel ADAR substrates. Mutant animals will also be assessed for changes in susceptibility to kainate-induced seizures and excitotoxic injury as well as changes in central feeding behavior. 3) The functional consequences of recently identifed editing events within Alu repetitive elements in 3'-untranslated regions (3'-UTR) of human RNAs will be assessed in relation to alterations in RNA stability and nuclear retention as a mechanism to modulate protein expression. It is anticipated that these studies will not only serve to define the functional consequences of A-to-l modification in non-coding regions of mRNAs, but also provide new insights concerning the physiological relevance of cellular processes modulating ADAR2 expression and their relationship to the function of neurotransmitter receptors involved in seizure susceptibility, energy balance and other aspects of CNS function.