RNA editing-mediated modulation of serotonin 2C receptor protein expression

描述（由申请人提供）：编码5 -羟色胺（5HT2C）受体2c亚型的转录本可以被多达5种腺苷-肌苷RNA编辑事件修饰，产生多达24种不同受体：g蛋白偶联功效和组成活性的蛋白质亚型。在与自杀相关的焦虑、精神分裂症和抑郁症等情感性疾病中，已经发现了5HT2C mrna编辑的变异。在最近的研究中，我们证明了仅表达5HT2C受体完全编辑亚型的转基因小鼠显示出与Prader-Willi综合征一致的表型变化，Prader-Willi综合征是一种人类发育障碍，其特征是初始发育失败、认知缺陷、躯体生长减少、新生儿肌肉张力降低和与病态肥胖相关的嗜食。有趣的是，与野生型动物相比，这些突变小鼠的5HT2C受体密度增加了前所未有的40至70倍，但稳态mRNA水平保持不变。因此，RNA编辑通过未知的转录后机制对5HT2C蛋白的表达产生显著影响。本提案的目的是研究基于RNA编辑的5HT2C受体密度改变的分子机制。在第一个目标中，5HT2C编辑对受体稳定性的影响将通过体内和体外模型系统来确定。编辑对5HT2C运输和/或配体激活以及随后受体稳定性的影响也将被检查。在目标2中，将研究未编辑和完全编辑的5HT2C亚型之间翻译的潜在差异。此外，MBII-52（一种可以影响5HT2C选择性剪接的小核仁RNA (snoRNA)）在编辑和未编辑的5HT2C mrna上的翻译作用将被研究。目的3是对全鼠脑中5HT2C蛋白相对异构体水平的定量分析。预计这些研究将为与5HT2C功能改变相关的人类疾病提供新的见解，其中关于编辑受体同种型表达的推断仅基于mRNA的分析。

DESCRIPTION (provided by applicant): Transcripts encoding the 2C-subtype of serotonin (5HT2C) receptor can be modified by up to five adenosine-to- inosine RNA editing events, generating as many as 24 protein isoforms that differ in their receptor:G-protein coupling efficacy and constitutive activity. Variations in the editing of 5HT2C mRNAs have been identified in affective disorders including anxiety, schizophrenia and depression associated with suicide. In recent work, we demonstrate that genetically-modified mice solely expressing the fully edited isoform of the 5HT2C receptor display phenotypic changes consistent with Prader-Willi Syndrome, a human developmental disorder characterized by an initial failure to thrive, cognitive deficits, decreased somatic growth, neonatal muscular hypotonia, and hyperphagia associated with morbid obesity. Interestingly, these mutant mice exhibit an unprecedented 40- to 70-fold increase in 5HT2C receptor density compared to wild-type animals, yet the steady-state mRNA levels remain unchanged. Thus, RNA editing has dramatic consequences on the expression of 5HT2C protein through uncharacterized post-transcriptional mechanism(s). The objectives of this proposal are to investigate the molecular mechanism(s) underlying RNA editing-based alterations in 5HT2C receptor density. In the first aim, the effects of 5HT2C editing on receptor stability will be determined using both in vivo and in vitro model systems. The effects of editing on 5HT2C trafficking and/or ligand activation and subsequent stability of the receptor also will be examined. In aim 2, potential differences in translation among non-edited and fully edited 5HT2C isoforms will be investigated. Furthermore, translation effects of MBII-52, a small nucleolar RNA (snoRNA) that can affect 5HT2C alternative splicing, will be investigated on both edited and non-edited 5HT2C mRNAs. Aim 3 represents a quantitative analysis of relative 5HT2C protein isoform levels in whole mouse brain. It is anticipated that these studies will provide novel insights into human disorders related to altered 5HT2C function, where inferences about the expression of edited receptor isoforms have been based solely upon analyses of mRNA. PUBLIC HEALTH RELEVANCE: This research plan will increase our understanding of the functional diversity and regulatory circuits of the serotonin 2C receptor (5HT2C) in the mammalian nervous system. Our novel findings have the potential to redefine research, diagnosis and treatment of 5HT2C-related disorders including schizophrenia, depression, anxiety and addiction.