EDITING OF PRE MRNA IN NEUROLOGICAL CELL LINES

描述(摘自申请者摘要)：ADAR2是一种腺苷脱氨酶 作用于RNA，即一种能使特定腺嘌呤(A)脱氨的RNA编辑酶 特定前信使核糖核酸中的残基产生肌苷(I)。因为核糖体 将肌苷读作鸟苷(G)，这些脱氨基导致密码子改变 这可能会导致相关的氨基酸序列发生变化 蛋白。生物上重要的转录本经历这种脱氨基作用 包括几个谷氨酸受体亚基和一种5-羟色胺前mRNAs 受体。在许多情况下，氨基酸的变化对 蛋白质的功能。脱氨的频率是由发育调节的。 而且很少达到100%。因此，这种类型的RNA编辑是一种机制 创造微妙而关键的蛋白质序列多样性。测量 脑组织中肌苷含量及ADAR2的表达水平 地区表明，还有许多额外的、尚未确定的ADAR2 底物。鉴于谷氨酸和5-羟色胺所起的关键作用 神经传递中的受体，大脑中信使核糖核酸编辑的整体作用 发展和作用可能是深远的。更改或修改 由多态或突变引起的编辑模式将是很好的候选 牵涉到神经异常。这一试点项目的目标是 评估ADAR2信使核糖核酸编辑的普遍性和相关性 表达的新底物的鉴定和特性 神经学来源的人类细胞系。为此，我们将使用一种新的程序 从人神经母细胞瘤和NT2中分离含肌苷的mRNA分子 转化细胞系稳定过表达人ADAR2基因。 含有肌苷的分子将被克隆、测序并用RT-PCR法进行分析 从A到I的模式变化。每个ADAR2底物都将在人体内被映射 还有老鼠。定义用于RNA编辑的新底物将定义新的来源 蛋白质多样性和基因表达调控，并将识别新的 神经疾病和发育异常的候选人。

DESCRIPTION (From the Applicant's Abstract): ADAR2 is an Adenosine Deaminase that acts on RNA, i.e. an RNA editase that can deaminate specific adenine (A) residues in specific pre-mRNAs to produce inosines (I). Because the ribosome reads an inosine as a guanosine (G), these deaminations cause codon changes that can result in changes in the amino acid sequence of the associated protein. Biologically important transcripts that undergo such deaminations include pre-mRNAs for several glutamate receptor subunits and for a serotonin receptor. In many of these cases the amino acid change has profound effects on the protein function. The frequency of deamination is developmentally regulated and rarely reaches 100 percent. Thus, this type of RNA editing is a mechanism for creating subtle and critical protein sequence diversity. Measurements of the inosine content of brain mRNA and the expression level of ADAR2 in brain regions suggest that there are numerous additional, as yet unidentified, ADAR2 substrates. Given the critical roles played by glutamate and serotonin receptors in neurotransmission, the overall role of mRNA editing in brain development and function may be profound. Alterations or modifications of editing patterns caused by polymorphism or mutation will be good candidates for involvement in neurological abnormalities. The goal of this pilot project is to evaluate the prevalence and relevance of ADAR2 mRNA editing by the identification and characterization of novel substrates expressed in neurologically derived human cell lines. To do this we will use a new procedure to isolate inosine-containing mRNA molecules from human neuroblastoma and NT2 cell lines transformed to stably overexpress human ADAR2 cDNA. Inosine-containing molecules will be cloned, sequenced, and analyzed by RT-PCR for patterns in A to I changes. Each ADAR2 substrate will be mapped in human and mouse. Definition of new substrates for RNA editing will define new sources of protein diversity and regulation of gene expression, and will identify new candidates for neurological disease and developmental anomalies.