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

亨廷顿病蛋白在转录后基因沉默中的作用

• 批准号: 8097418
• 负责人: NAOKO TANESE
• 金额: $ 32.61万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8097418
• 关键词: Address; Affect; Age; Animals; Binding; Biological Assay; Biological Process; Brain Diseases; Cell Line; Cell physiology; Cells; Codon Nucleotides; Collaborations; Complex; Corpus striatum structure; Cytoplasmic Granules; Cytoplasmic Structures; Data; Disease; Drosophila genus; Employee Strikes; Epitopes; Exhibits; Gene Expression; Gene Silencing; Gene Silencing Pathway; Health; Hela Cells; Huntington Disease; Immunofluorescence Immunologic; Impaired cognition; Inherited; Kinetics; Knock-out; Length; Maps; Measures; Mediating; Messenger RNA; MicroRNAs; Molecular; Motor Neurons; Mus; Neurodegenerative Disorders; Neurons; Pathway interactions; Patients; Play; Post-Transcriptional Regulation; Process; Proteins; RNA; RNA Interference; Regulation; Reporter; Reporting; Repression; Role; Small RNA; Specificity; Staging; Symptoms; Testing; Therapeutic Intervention; Translational Repression; Triplet Multiple Birth; Two-Hybrid System Techniques; Yeasts; base; cDNA Arrays; driving force; embryonic stem cell; gain of function; gene repression; human Huntingtin protein; insight; mRNA Decay; mRNA Surveillance; mRNA Transcript Degradation; middle age; mutant; novel; polyglutamine; research study; yeast two hybrid system

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Huntington's disease (HD) is a devastating disease that strikes patients in mid-life with symptoms such as motor neuron dysfunction, cognitive and psychiatric disturbances that worsen with age. There is no cure for HD and currently available therapies are of limited use. Better understanding of the functions of the disease-causing huntingtin protein and the pathogenic mechanisms involved in the early stages of HD would permit identification of new targets for therapeutic intervention. Furthermore, studies suggest that other neurodegenerative disorders caused by poly-glutamine expansion may share similar disease mechanisms. Thus, studies on HD will likely contribute to the molecular understanding of many diseases of the brain.

描述(申请人提供)：亨廷顿病(HD)是一种显性常染色体退行性疾病，由亨廷顿蛋白(Htt)氨基末端的聚谷氨酰胺(PolyQ)链扩张引起，其细胞功能仍存在争议。为了深入了解Htt的功能，揭示HD的潜在致病机制，从HeLa细胞中纯化了Htt表位标记的Htt蛋白，并鉴定了ArgAerte(AGO)蛋白为Htt相互作用蛋白。AGOS被证明部分定位于离散的细胞质内，称为P(加工)体。参与小RNA介导的基因沉默、翻译抑制、信使核糖核酸监视和信使核糖核酸降解的蛋白质在P小体上与其结合的mRNAs一起积累，在那里它们的命运被决定。共定位研究表明Htt存在于P小体中。此外，Htt的缺失显示了RNAi介导的基因沉默，表明正常的Htt参与了基因表达的转录后调节。对表达内源性突变体Htt的小鼠纹状体细胞的分析显示，与野生型纹状体细胞相比，P小体较少，基因沉默活性降低。这些数据表明，先前报道的HD转录失控可能部分归因于突变的Htt在转录后过程中的作用。Htt与Ago2的关联以及Htt对P小体的定位为Htt与转录后控制mRNA丰度和P小体完整性提供了新的令人信服的证据。假设Htt作为一种AGO辅助因子，通过RNAi介导的机制或与P小体相关的翻译抑制/mRNA衰退途径来调节蛋白质水平。神经元RNA颗粒已被描述为参与神经元mRNAs的运输和翻译控制。最近的一项研究报道了果蝇神经元颗粒中存在P-小体蛋白。人们很容易推测Htt参与了神经元RNA颗粒的功能，这种功能可能被突变的Htt干扰，有助于HD神经元的特异性。在这项提案中，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基因敲除或基因敲除细胞中进行microRNA报告分析和功能拴系分析。P小体在纹状体细胞以及表达内源性wt或突变型htt的原代神经元中的分布将通过免疫荧光来确定。纯化数据表明，野生型Htt优先与Ago2结合，而突变型Htt与参与基因沉默的P小体的另一组分TNRC6B结合。推测突变体Htt和TNRC6B的富含Q结构域与P小体功能的改变有关。Htt-TNRC6B的相互作用将使用酵母双杂交系统进行检测。与Htt-Ago2复合体相关的mRNAs和miRNAs将通过基因芯片和TaqMan miRNA分析进行纯化和鉴定，并将研究它们作为Htt新靶点的生物学功能。这些实验应该为Htt在转录后途径中以前未被描述的角色提供新的见解，并有助于揭示导致HD的新的致病机制。与公共卫生相关：亨廷顿病(HD)是一种毁灭性的疾病，患者在中年时会出现运动神经元功能障碍、认知和精神障碍等症状，并随着年龄的增长而恶化。目前还没有治愈HD的方法，目前可用的治疗方法也有限。更好地了解致病亨廷顿蛋白的功能和HD早期阶段涉及的致病机制，将有助于确定治疗干预的新靶点。此外，研究表明，由聚谷氨酰胺扩张引起的其他神经退行性疾病可能具有类似的疾病机制。因此，对HD的研究可能有助于从分子上理解许多脑部疾病。