RNA Editing-Mediated Regulation of Serotonin 2C Receptor Density

 描述(申请人提供)：5-羟色胺受体的2C亚型(5HT2C)在大脑中广泛表达，并与几种神经精神障碍有关，包括严重抑郁障碍、心境恶劣、强迫症、焦虑、双相情感障碍和精神分裂症。编码5HT2C受体的转录本可以通过最多五次腺苷到肌苷的编辑事件进行差异修饰，这一过程负责在受体的第二细胞内环中差异多达三个氨基酸的多达24个蛋白质亚型的细胞特异性表达，该区域对于G蛋白偶联效果至关重要。在体外，完全编辑的5HT2C亚型(5HT2C-VGV)与基因组编码的亚型(5HT2C-INI)相比，结构活性降低，亚细胞定位改变。因此，5HT2C编辑可能代表了一种关键的调控机制，通过该机制，神经元通过改变受体：G蛋白相互作用的有效性和特异性来调节其对细胞外信号变化的反应。此外，在被诊断为精神分裂症和双相情感障碍的患者中，在有严重抑郁症病史的自杀受害者中，以及在抗抑郁和抗精神病药物治疗的反应中，观察到了5HT2C编辑的变化，这表明5HT2C转录本的不当编辑可能是神经精神疾病的一个促成因素。这项拟议研究的长期目标是确定调控5HT2C表达和信号的细胞机制，以及5HT2C编辑和神经病理之间的可能关系。最近的研究表明，单独表达完全编辑的5HT2C受体亚型(5HT2C-VGV)的转基因小鼠，其受体密度史无前例地增加了40-70倍，而稳态5HT2C mRNA没有同时发生变化。因此，RNA编辑通过未知的转录后机制影响5HT2C蛋白的表达(S)。为了评估在野生型小鼠中编辑的5HT2C转录本和它们的编码蛋白产物之间是否存在差异，将使用亲和纯化方法分离5HT2C受体，然后进行质谱学蛋白质组学分析以定量5HT2C蛋白亚型的相对表达水平。最近开发的一种基于CRISPR/CAS的方法将被用于产生突变小鼠，在该突变小鼠中，将表位标签(S)引入内源性5HT2C基因座，从而能够更有效地纯化5HT2C蛋白，用于随后比较不同脑区的基因和蛋白质亚型分布。识别5HT2C RNA和蛋白质异构体表达之间的差异将对人类研究5HT2C RNA编辑中与疾病相关的变化具有重要意义，在5HT2C RNA编辑中，关于受体异构体表达和功能的推断仅基于编辑的mRNA分布。此外，这些发现将增加编辑其他mRNA转录本可能类似地影响后续蛋白质表达水平的可能性，从而扩大这种RNA加工事件在哺乳动物生物学中的作用。

 DESCRIPTION (provided by applicant): The 2C subtype of serotonin receptor (5HT2C) is widely expressed in the brain and has been implicated in several neuropsychiatric disorders, including major depressive disorder, dysthymia, obsessive-compulsive disorder, anxiety, bipolar disorder, and schizophrenia. Transcripts encoding the 5HT2C receptor can be differentially modified by up to five adenosine-to-inosine editing events, a process responsible for the cell-specific expression of as many as 24 protein isoforms that differ by up to three amino acids within the second intracellular loop of the receptor, a region essential for G protein coupling efficacy. In vitro, the fully edited 5HT2C isoform (5HT2C-VGV) exhibits reduced constitutive activity and altered subcellular localization in comparison to the genomically-encoded isoform (5HT2C-INI). Thus, 5HT2C editing may represent a critical regulatory mechanism by which neurons modulate their response to changing extracellular signals by altering the efficacy and specificity of receptor:G protein interactions. Moreover, alterations in 5HT2C editing have been observed in patients diagnosed with schizophrenia and bipolar disorder, in suicide victims with a history of major depression, and in response to antidepressant and antipsychotic treatments, suggesting that improper editing of 5HT2C transcripts may be a contributing factor in neuropsychiatric illness. The long term objectives of the proposed research are to define the cellular mechanisms that regulate 5HT2C expression and signaling, as well as possible relation- ships between 5HT2C editing and neuropathologies. Recent studies have revealed that genetically modified mice solely expressing the fully edited 5HT2C receptor isoform (5HT2C-VGV) produce an unprecedented 40- to 70-fold increase in receptor density without a concurrent change in steady-state 5HT2C mRNA. Thus, RNA editing has dramatic consequences on the expression of 5HT2C protein through uncharacterized post- transcriptional mechanism(s). To evaluate whether a disparity exists between edited 5HT2C transcripts and their encoded protein products in wild-type mice, affinity purification methods wil be used to isolate 5HT2C receptors, followed by a mass spectrometric proteomic analysis to quantify the relative expression levels of 5HT2C protein isoforms. A recently developed CRISPR/Cas-based approach will be used to generate mutant mice in which epitope tag(s) have been introduced into to the endogenous 5HT2C locus, thus enabling a more effective purification of 5HT2C protein for subsequent comparisons of mRNA and protein isoform distribution in discrete brain regions. Identification of disparities between 5HT2C RNA and protein isoform expression will have important implications for human studies of disease-related alterations in 5HT2C RNA editing, in which inferences about receptor isoform expression and function have been based solely upon edited mRNA distribution profiles. Furthermore, these findings will raise the possibility that editing of other mRNA transcripts may similarly affect subsequent protein expression levels, thus expanding the role for this RNA processing event in mammalian biology.