Molecular analysis of the genes involved in SCA8 ataxia

描述(研究人员摘要)：脊髓小脑性共济失调8型(SCA8) 由CTG在未翻译的内源性反义RNA中扩增引起的 与Kelch-like 1(KLHL1)基因重叠。KLHL1启动子与开放阅读 KLHL1反义转录本(KLHL1AS)是 也存在于小鼠体内。我们已经进行了初步的表征 KLHL1AS和KLHL1基因在人类和小鼠中都存在，但我们在这一点上 关于这些基因的一些基本问题：1)什么是正常的 进化保守的KLHL1-反义RNA的功能？，2)什么作用 KLHL1蛋白在表达KLHL1蛋白的神经元中起作用吗？ CTG扩增如何影响KLHL1AS RNA和KLHL1蛋白，以及 这些效应解释了SCA8患者的神经退行性变吗？ 我们到目前为止所获得的数据使我们能够做出明智的假设 针对这些问题，设计了一些活体实验系统 测试、改进并在必要时重新表述这些假设。这个 KLHL1mRNA和KLHL1AS转录本的转录组织 提示KLHL1AS很可能是KLHL1表达的调节因子。基于 KLHL1与其他神经元特异性海带样蛋白的同源性 对该蛋白的初步鉴定，我们推测KLHL1可能发挥了作用 在组织肌动蛋白纤维中的作用，帮助产生和维持轴突 和树枝状结构。我们不能预先预测SCA8 CTG扩展可能会产生什么影响 KLHL1/KLHL1AS基因系统。然而，这两个基因都是特定的 在小脑中表达，所以这些转录产物很可能是 直接或通过调节这种扩张的致病作用 改变了反义相互作用。 我们将对编码的BAC克隆执行多次迭代修改 人KLHLAS基因和KLHL1基因前两个外显子在大肠杆菌中的同源克隆 重组。然后我们将把修改后的BAC克隆引入组织中 培养细胞直接确定KLHL1和KLHL1AS如何相互作用和测量 这些相互作用对KLHL1的表达水平有什么影响。替换 最后一个KLHL1AS外显子中的CTG重复与这些克隆中的扩展重复将 还允许我们测试SCA8扩展如何影响KLHL1AS和KLHL1，以及如何 它改变了这些基因之间的相互作用。尽管我们将继续 描述结构、蛋白质相互作用以及细胞和亚细胞 KIHI1蛋白的定位，我们将直接测试这一作用 通过产生KLHL1基因参与正常神经元发育和功能的蛋白质 在老鼠体内进行了基因敲除。我们设计的淘汰赛策略将以一种 允许我们1)研究破坏KLHL1基因对 特定于组织和特定于时间的方式，以及2)决定 KLHL1AS转录水平的改变对KLHL1的表达和功能有影响。

DESCRIPTION (Investigator's abstract): Spinocerebellar ataxia type 8 (SCA8) is caused by a CTG expansion in an untranslated, endogenous antisense RNA that overlaps the Kelch-like 1 (KLHL1) gene. The KLHL1 promoter and open-reading frame are conserved in mouse, and a KLHL1.antisense transcript (KLHL1AS) is present in mouse as well. We have performed initial characterization of the KLHL1AS and KLHL1 genes in both man and mouse, but we are at this point left with some fundamental questions regarding these genes: 1) What is the normal function of the evolutionarily conserved KLHL1-antisense RNA?, 2) What role does the KLHL1 protein play in the neurons in which it is expressed?, and 3) How does the CTG expansion affect the KLHL1AS RNA and KLHL1 protein, and can these effects explain the neurodegeneration seen in SCA8 patients? The data we have obtained to date have allowed us to make informed hypotheses concerning these questions and to design some in vivo experimental systems to test, refine, and if necessary reformulate these hypotheses. The transcriptional organization of the KLHL1 mRNA and the KLHL1AS transcript suggests that KLHL1AS is most likely a regulator of KLHL1 expression. Based on the homology of KLHL1 to other neuron-specific kelch-like proteins and on our preliminary characterization of this protein, we speculate that KLHL1 may play a role in organizing the actin fibers that help to generate and maintain axons and dendrites. We cannot predict a priori how the SCA8 CTG expansion may affect the KLHL1/KLHL1AS gene system. Both of these genes, however, are specifically expressed in the cerebellum, and so these transcripts are likely candidates for mediating the pathogenic effect of this expansion either directly or through altered antisense interactions. We will perform multiple, iterative modifications of a BAC clone encoding the human KLHLAS gene and the first two exons of KLHL1 in E. coli using homologous recombination. We will then introduce the modified BAC clones into tissue culture cel Is to directly determine how KLHL1 and KLHL1AS interact and measure what effect these interactions have on KLHL1 expression levels. Replacing the CTG repeat in the last KLHL1AS exon with expanded repeats in these clones will also allow us to test how the SCA8 expansion affects KLHL1AS and KLHL1, and how it alters the interactions between these genes. Although we will continue to characterize the structure, protein interactions and cellular and subcellular localization of the KIHI1 protein, we will directly test the role of this protein in normal neuronal development and function by generating a KLHL1 gene knockout in mouse. We have designed our knockout strategy in a way that will allow us to both 1) study the effects of disrupting the KLHL1 gene in a tissue-specific and temporal-specific manner, and 2) determine the effect that altered levels of KLHL1AS transcription has on KLHL1 expression and function.