REGULATION OF POST-TRANSCRIPTIONAL GENE SILENCING

描述（来自应用程序）：转录后基因沉默（PTGS） 是一种古老的真核生物调控机制， 序列被锁定并摧毁虽然PTGS发生在不同的生物体中， 该通路的细胞组分及其调节不好 明白植物马铃薯Y病毒属的辅助组分蛋白酶（HG-Pro）具有 抑制植物中的PTGS。在酵母中使用HG-Pro作为诱饵 利用双杂交系统，我们鉴定了一个新的钙调素相关蛋白 （称为rgsCaM），其与PTGS的这种病毒抑制剂相互作用。rgs-CaM， 像HG-Pro本身一样，抑制PTGS的启动和维持。我们 假设HC-Pro抑制PTGS是通过其相互作用介导的 rgs-CaM在这里，我们建议利用HC-Pro和rgs-CaM作为工具， 植物沉默的机制该提案的前四个目标侧重于 研究rgs-CaM在PTGS中的作用，鉴定蛋白质的结构域， 抑制PTGS以及参与rgs-CaM/HC-Pro相互作用的那些。 将使用几种方法来干扰rgs-CaM的表达， 这些操作对PTGS的影响以及HC-Pro对其的抑制将被 化验。第三个目标是解决机械问题，确定如何和 其中HC-Pro和rgs-CaM作用于PTGS中的已知步骤。最后两 目的是关注其他植物蛋白在HC-Pro介导的 抑制PTGS。我们将继续调查其他三个 HC-Pro相互作用蛋白使用的方法，成功地确定 rgs-CaM作为PTGS的调节剂。基因阵列技术将用于识别 在PTGS应答或HC-Pro逆转中受到调控的基因。 最后，我们将利用模式遗传生物拟南芥 筛选干扰PTGS抑制的突变体， HC-Pro。植物沉默系统作为一个模型，以了解类似的 在动物中的途径，并有可能被用于基因治疗 基因表达的应用和操作。鉴于抗病毒的性质， 基因沉默，了解植物中的PTGS可以很好地导致 开发人类抗病毒策略。

DESCRIPTION (from the application): Post-transcriptional gene silencing (PTGS) is an ancient eukaryotic regulatory mechanism in which a particular RNA sequence is targeted and destroyed. Although PTGS occurs in diverse organisms, the cellular components of the pathway and its regulation are not well understood. The helper component proteinase (HG-Pro) of plant potyviruses has been shown to suppress PTGS in plants. Using HG-Pro as bait in a yeast two-hybrid system, we have identified a novel calmodulin-related protein (termed rgsCaM) that interacts with this viral suppressor of PTGS. rgs-CaM, like HG-Pro itself, suppresses both initiation and maintenance of PTGS. Our hypothesis is that HC-Pro-suppression of PTGS is mediated by its interaction with rgs-CaM. Here we propose to exploit HC-Pro and rgs-CaM as tools to dissect the mechanism of silencing in plants. The first four aims of the proposal focus on the role of rgs-CaM in PTGS, identifying domains of the protein required to suppress PTGS as well as those involved in the rgs-CaM/HC-Pro interaction. Several approaches will be used to interfere with expression of rgs-CaM and the effect of these manipulations on PTGS and its suppression by HC-Pro will be assayed. The third aim addresses mechanistic questions, determining how and where HC-Pro and rgs-CaM act with regard to known steps in PTGS. The last two aims focus on the role of other plant proteins in the HC-Pro-mediated suppression of PTGS. We will extend investigations of three other HC-Pro-interacting proteins using the approaches that successfully identified rgs-CaM as a regulator of PTGS. Gene array technology will be used to identify genes that are regulated in response to PTGS or its reversal by HC-Pro. Finally, we will exploit the model genetic organism Arabidopsis thalianato screen for mutants that interfere with suppression of PTGS in response to HC-Pro. The plant silencing system serves as a model to understand similar pathways in animals, and has potential to be exploited for gene therapy applications and manipulation of gene expression. Given the antiviral nature of gene silencing in plants, understanding PTGS in plants could well lead to the development of antiviral strategies in humans.