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: 7730432
• 负责人: NAOKO TANESE
• 金额: $ 35.84万
• 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/7730432
• 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; 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; public health relevance; 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（由申请人提供）：亨廷顿病（HD）是一种显性常染色体神经退行性疾病，由亨廷顿蛋白（Htt）氨基末端的多谷氨酰胺（polyQ）通道扩大引起，其细胞功能仍有争议。为了深入了解Htt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，并鉴定了Argonaute （Ago）蛋白为Htt相互作用蛋白。艾格斯已经被证明部分地定位于被称为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降解的蛋白质与它们结合的mRNA一起在p小体中积累，在那里它们的命运被决定。共定位研究表明Htt存在于p体中。此外，Htt的缺失显示rnai介导的基因沉默受损，这表明正常的Htt有助于基因表达的转录后调控。对表达内源性突变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，先前报道的HD的转录失调可能部分归因于突变体Htt在转录后过程中的作用。Htt与Ago2的关联以及Htt在p -小体上的定位提供了新的令人信服的证据，证明Htt与mRNA丰度和p -小体完整性的转录后控制有关。据推测，Htt作为Ago辅助因子通过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。神经元RNA颗粒已被描述为参与转运和翻译控制神经元mrna。最近的一项研究报道了果蝇神经元颗粒中p体蛋白的存在。我们很容易推测，Htt参与了神经元RNA颗粒的功能，而这种功能可能被突变的Htt干扰，从而导致了HD中神经元的特异性。在本提案中，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敲除或敲除细胞中进行microRNA报告细胞测定和功能栓系测定。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p小体的分布将通过免疫荧光测定。纯化数据表明，野生型Htt优先与Ago2结合，而突变型Htt与TNRC6B结合，TNRC6B是参与基因沉默的p小体的另一个组分。据推测，突变体Htt和TNRC6B的富q结构域有助于p体功能的改变。Htt-TNRC6B相互作用将使用酵母双杂交系统进行检测。Htt- ago2复合体的相关mrna和miRNA将通过cDNA微阵列和TaqMan miRNA检测进行纯化和鉴定，并研究其作为Htt新靶点的生物学功能。这些实验应该为Htt在转录后通路中以前未被描述的作用提供新的见解，并有助于揭示HD的新致病机制。公共卫生相关性：亨廷顿舞蹈病（HD）是一种毁灭性的疾病，中年患者会出现运动神经元功能障碍、认知和精神障碍等症状，这些症状会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作用有限。更好地了解致病的亨廷顿蛋白的功能以及HD早期阶段的致病机制将有助于确定新的治疗干预靶点。此外，研究表明，由多谷氨酰胺扩增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的研究可能有助于对许多脑部疾病的分子理解。