Molecular analysis of the genes involved in SCA8 ataxia

描述（研究者摘要）：脊髓小脑性共济失调8型（SCA 8）是 由未翻译的内源性反义RNA中的CTG扩增引起， 与Kelch样1（KLHL 1）基因重叠。KLHL 1启动子和开放阅读 框架在小鼠中是保守的，并且KLHL1.antisense transcript（KLHL 1AS）在小鼠中是保守的。 在老鼠身上也有。我们已经进行了初步表征的 KLHL 1AS和KLHL 1基因在人类和小鼠中，但我们在这一点上离开了 关于这些基因的一些基本问题：1）什么是正常的 进化上保守的KLHL 1反义RNA的功能，2)什么角色 KLHL 1蛋白在表达它的神经元中发挥作用吗？和3） CTG扩增如何影响KLHL 1AS RNA和KLHL 1蛋白， 这些效应解释了SCA 8患者的神经变性？ 迄今为止我们获得的数据使我们能够做出明智的假设 并设计了一些体内实验系统， 测试、提炼并在必要时重新表述这些假设。的 KLHL 1 mRNA和KLHL 1AS转录本的转录组织 提示KLHL 1AS很可能是KLHL 1表达的调节因子。基于 KLHL 1与其他神经元特异性kelch样蛋白的同源性， 初步表征这种蛋白质，我们推测KLHL 1可能发挥作用， 在组织肌动蛋白纤维，帮助产生和维持轴突的作用 和树突。我们不能先验地预测SCA 8 CTG扩展可能如何影响 KLHL 1/KLHL 1AS基因系统。然而，这两个基因都是特定的 在小脑中表达，因此这些转录本可能是 直接或通过介导这种扩张的致病作用 改变反义相互作用。 我们将对BAC克隆进行多次迭代修改， 人KLHLAS基因和KLHL 1基因前两个外显子在大肠杆菌中的表达。大肠杆菌使用同源 重组然后我们将修改后的BAC克隆导入组织中， 培养细胞直接确定KLHL 1和KLHL 1AS如何相互作用和测量 这些相互作用对KLHL 1表达水平的影响。更换 最后一个KLHL 1AS外显子中的CTG重复序列与这些克隆中的扩展重复序列将 也使我们能够测试SCA 8扩展如何影响KLHL 1AS和KLHL 1，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 表征结构、蛋白质相互作用以及细胞和亚细胞 KIHI 1蛋白的定位，我们将直接测试这种作用， 通过产生KLHL 1基因在正常神经元发育和功能中的蛋白质 敲除小鼠。我们设计的淘汰赛策略 使我们能够1）研究破坏KLHL 1基因在一个细胞中的影响， 组织特异性和时间特异性方式，以及2）确定 KLHL 1AS转录水平的改变对KLHL 1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.