Role of the Huntington's Disease Protein in Post-Transcriptional Gene Silencing

亨廷顿病（HD）是由亨廷顿蛋白（Htt）氨基端的多聚谷氨酰胺（polyQ）链扩展引起的显性常染色体神经退行性疾病，其细胞功能仍存在争议。为了深入了解Htt的功能并揭示潜在的HD致病机制，从HeLa细胞中纯化了表位标记的Htt，并鉴定Argonaute（Ago）蛋白为Htt相互作用蛋白。Agos已被部分显示定位于称为P（加工）体的离散细胞质灶。参与小RNA介导的基因沉默、翻译抑制、mRNA监视和mRNA降解的蛋白质与其结合的mRNA一起在P体积累，在P体处决定其命运。共定位研究表明Htt存在于P体中。此外，耗尽的Htt显示受损的RNAi介导的基因沉默，表明正常的Htt有助于基因表达的转录后调控。表达内源性突变Htt的小鼠纹状体细胞的分析显示，与野生型相比，P体较少，基因沉默活性降低。这些数据表明，以前报道的转录失调HD可能是由于部分突变体Htt的作用，在转录后过程。Htt与Ago 2的关联和Htt定位于P体提供了新的和令人信服的证据，将Htt与mRNA丰度和P体完整性的转录后控制联系起来。据推测，Htt作为Ago辅助因子的功能是通过RNAi介导的机制或与P体相关的翻译抑制/mRNA衰变途径调节蛋白质水平所必需的。神经元RNA颗粒已被描述为参与神经元mRNA的转运和翻译控制。最近的一项研究报道了果蝇神经元颗粒中存在P体蛋白。这是诱人的推测，Htt参与神经元RNA颗粒功能，这可能是由突变Htt扰动，有助于在HD神经元的特异性。在这个提议中，野生型和突变型Htt在转录后过程中的作用将被研究，重点是RNA介导的基因沉默。将在小鼠纹状体细胞和Htt敲低或敲除细胞中进行microRNA报告基因测定和功能性拴系测定。将通过免疫荧光测定纹状体细胞以及表达内源性wt或突变体Htt的原代神经元中P体的分布。纯化数据表明，野生型Htt优先与Ago 2相关联，而突变型Htt与TNRC 6 B相关联，TNRC 6 B是参与基因沉默的P体的另一种组分。假设突变体Htt和TNRC 6 B的富Q结构域有助于P体功能的改变。将使用酵母双杂交系统检查Htt-TNRC 6 B相互作用。通过cDNA微阵列和TaqMan miRNA检测技术对Htt-Ago 2复合物相关的mRNA和miRNA进行纯化和鉴定，并研究其作为Htt新靶点的生物学功能。这些实验应该提供新的见解，以前未知的作用Htt在转录后通路，并有助于发现新的致病机制，有助于HD。

Huntington's disease (HD) is a dominant autosomal neurodegenerative disorder caused by an expanded poly-glutamine (polyQ) tract within the amino-terminus of the huntingtin (Htt) protein, whose cellular function remains controversial. To gain insight into Htt function and uncover potential HD pathogenic mechanisms, epitope-tagged Htt was purified from HeLa cells and Argonaute (Ago) proteins were identified as Htt-interacting proteins. Agos have been shown in part to localize to discrete cytoplasmic foci known as P (processing)-bodies. Proteins involved in small RNA-mediated gene silencing, translational repression, mRNA surveillance, and mRNA degradation accumulate with their bound mRNAs at P-bodies, where their destiny is determined. Co-localization studies demonstrated Htt to be present in P-bodies. Furthermore, depletion of Htt showed compromised RNAi-mediated gene silencing, indicating that normal Htt contributes to post-transcriptional regulation of gene expression. Analysis of mouse striatal cells expressing endogenous mutant Htt showed fewer P-bodies and exhibited reduced gene silencing activity compared with their wild-type counterparts. These data suggest that the previously reported transcriptional deregulation in HD may be attributed in part to mutant Htt's role in post-transcriptional processes. The association of Htt with Ago2 and the localization of Htt to P-bodies provide new and compelling evidence connecting Htt with the post-transcriptional control of mRNA abundance and P-body integrity. It is hypothesized that Htt functions as an Ago accessory factor necessary for the regulation of protein levels through RNAi-mediated mechanisms or translational repression/mRNA decay pathways associated with P-bodies. Neuronal RNA granules have been described to be involved in transport and translational control of neuronal mRNAs. A recent study reported the presence of P-body proteins in neuronal granules in Drosophila. It is tempting to speculate that Htt is involved in neuronal RNA granule function, which may be perturbed by mutant Htt, contributing to specificity for neurons in HD. In this proposal the role of wild-type and mutant Htt in post-transcriptional processes will be investigated with a focus on RNA- mediated gene silencing. microRNA reporter assays and functional tethering assays in mouse striatal cells and Htt knockdown or knockout cells will be performed. The distribution of P-bodies in striatal cells as well as in primary neurons expressing endogenous wt or mutant Htt will be determined by immunofluorescence. The purification data indicates that wild-type Htt preferentially associates with Ago2 while mutant Htt associates with TNRC6B, another component of P-bodies involved in gene silencing. It is hypothesized that the Q-rich domains of mutant Htt and TNRC6B contribute to alterations in P-body function. The Htt-TNRC6B interaction will be examined using the yeast two-hybrid system. mRNAs and miRNAs associated with Htt-Ago2 complex will be purified and identified by cDNA microarrays and TaqMan miRNA assays and their biological function as new targets of Htt will be investigated. These experiments should provide new insights to previously uncharacterized role of Htt in post-transcriptional pathways and help to uncover new pathogenic mechanisms that contribute to HD.