EDITING OF PRE MRNA IN NEUROLOGICAL CELL LINES

描述（来自申请人的摘要）：ADAR 2是腺苷脱氨酶 作用于RNA，即可以使特定腺嘌呤脱氨基的RNA编辑酶（A） 在特定的前体mRNA的残基产生肌苷（I）。因为核糖体 将肌苷读作鸟苷（G），这些脱氨基作用导致密码子改变 这可能导致相关蛋白质的氨基酸序列发生变化， 蛋白生物学上重要的转录本， 包括几种谷氨酸受体亚基和5-羟色胺前体mRNA 受体的在许多这些情况下，氨基酸的变化有深远的影响， 蛋白质的功能。脱氨作用的频率是发育调节的 很少能达到100%。因此，这种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 脑组织肌苷含量和ADAR 2表达水平 区域表明，有许多额外的，尚未确定的，ADAR 2 印刷受体.考虑到谷氨酸和血清素的重要作用 受体在神经传递中的作用，mRNA编辑在大脑中的整体作用 发展和功能可能是深远的。改装或改动 由多态性或突变引起的编辑模式将是很好的候选者， 神经系统异常该试点项目的目标是 评估ADAR 2 mRNA编辑的患病率和相关性， 表达的新底物的鉴定和表征 神经衍生的人类细胞系。为了做到这一点，我们将使用一个新的程序 从人神经母细胞瘤和NT 2中分离含肌苷的mRNA分子， 转化细胞系以稳定过表达人ADAR 2 cDNA。 含肌苷的分子将被克隆，测序，并通过RT-PCR分析 从A到I的模式变化每个ADAR 2底物将在人体中定位。 和鼠标。定义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，并将确定新的 神经系统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.