Investigating the Mechanism and Effect of Disease-Associated Increases in the Huntingtin Long 3'UTR Isoform

研究亨廷顿蛋白长 3UTR 亚型与疾病相关的增加的机制和影响

• 批准号: 9047831
• 负责人: Lindsay Sara Romo
• 金额: $ 2.97万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-17 至 2019-09-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9047831
• 关键词: 3' Untranslated Regions; Affect; Alleles; Biological Assay; Bromine; CAG repeat; Cell Nucleus; Cell model; Cells; Cessation of life; Cognitive; Data; Disease; Effectiveness; Emotional; Environment; Exhibits; Exons; Fibroblasts; Fluorescent in Situ Hybridization; Genes; Genetic Transcription; Half-Life; Hour; Human; Huntington Disease; Label; Length; Measures; Mediating; Messenger RNA; Modeling; Motor; Mus; Mutation; Neurodegenerative Disorders; Neurons; Nuclear; Nucleotides; Open Reading Frames; Pathology; Patients; Physiologic pulse; Plasmids; Polyadenylation; Polyadenylation Pathway; Process; Protein Isoforms; Proteins; RNA; RNA Sequences; Relative (related person); Research; Research Personnel; Role; Sampling; Single Nucleotide Polymorphism; Small Interfering RNA; Small RNA; Specificity; Stable Disease; Testing; Therapeutic; Transfection; Translating; Translations; Trinucleotide Repeat Expansion; Untranslated Regions; Uridine; Wild Type Mouse; Work; actinomycin; deep sequencing; design; effective therapy; human Huntingtin protein; improved; insight; mRNA Stability; middle age; mouse model; mutant; neuronal survival; neurotoxic; polyadenylated messenger RNA; prevent; promoter; public health relevance; targeted treatment; transcriptome sequencing

 DESCRIPTION (provided by applicant): Huntington's disease is a devastating neurodegenerative disorder caused by translation of an expanded trinucleotide repeat in the huntingtin (HTT) gene into a neurotoxic protein. There is no effective treatment or cure, although researchers are developing small interfering RNAs to target and degrade mutant mRNA. Because the normal HTT protein is essential for neuronal survival, therapies aim to spare wild-type mRNA. It may be possible to specifically target the mutant mRNA if mutant and wild-type alleles are processed differently after transcription. Research indicates mutant and wild-type mRNA exhibit different stability and localization, but the mechanism is unknown. One possibility is mutant mRNA is processed into a different isoforms with different localization and stability. Mutant and wild-type HTT mRNA are present as two 3'UTR isoforms. Indeed, preliminary data suggest mutant mRNA is more likely than wild-type mRNA to be present as the longer isoform. The proposed work aims to determine the mechanism and impact of differences in mutant and wild-type HTT 3'UTR isoforms. Because the mutation is proximal to gene regions involved in alternative polyadenylation (APA), the expansion may disrupt mutant HTT APA and cause the shift to the longer isoform. If so, other mRNAs and the other allele should be processed normally. To determine if the change in isoform length is HTT-specific, the length of HTT and other APA mRNAs will be assessed in heterozygous human samples by qPCR and deep sequencing. To verify the expansion is responsible, isoform lengths will be measured by qPCR from a transfected plasmid with or without the expansion. To assess whether the HTT isoform length changes during or after APA, the length of HTT isoforms from the plasmid will be assessed right after or much after labeling newly transcribed mRNAs with a modified nucleotide. These studies are the focus of Specific Aim 1. The change in mutant HTT isoform length likely impacts downstream processing because the 3'UTR contains many motifs directing mRNA stability and localization. To test this, the decay and localization of mutant and wild-type HTT isoforms will be assayed in normal and disease cells by transcriptional arrest and qPCR (decay) or fluorescent in-situ hybridization (localization). To verify differences are due to the isoform length, isoform localization and decay will be assayed in cell models transfected with a single HTT isoform. These studies are the focus of Specific Aim 2. Together, these studies will elucidate differences in mutant and wild-type mRNA APA, decay, and localization. Allele-specific small RNAs could then be designed to target mRNA regions or cellular compartments unique to the mutant isoform, improving treatment of this devastating disease.

 描述（由申请人提供）：亨廷顿病是一种破坏性神经退行性疾病，由亨廷顿蛋白（HTT）基因中扩展的三核苷酸重复翻译成神经毒性蛋白引起。目前还没有有效的治疗或治愈方法，尽管研究人员正在开发小干扰RNA来靶向和降解突变的mRNA。由于正常的HTT蛋白对于神经元存活是必不可少的，因此治疗的目的是保留野生型mRNA。如果突变体和野生型等位基因在转录后被不同地加工，则可能特异性地靶向突变体mRNA。研究表明，突变型和野生型mRNA表现出不同的稳定性和定位，但其机制尚不清楚。一种可能性是突变mRNA被加工成具有不同定位和稳定性的不同同种型。突变体和野生型HTT mRNA以两种3'UTR同种型存在。事实上，初步数据表明突变mRNA比野生型mRNA更可能以较长的同种型存在。这项工作旨在确定突变型和野生型HTT 3'UTR亚型差异的机制和影响。由于突变接近参与选择性多聚腺苷酸化（阿帕）的基因区域，因此扩增可能会破坏突变HTT阿帕并导致转变为较长的同种型。如果是这样，其他mRNA和其他等位基因应该正常加工。为了确定亚型长度的变化是否具有HTT特异性，将通过qPCR和深度测序在杂合子人样本中评估HTT和其他阿帕mRNA的长度。为了验证扩增是否可靠，将通过qPCR从有或没有扩增的转染质粒中测量同种型长度。为了评估HTT同种型长度在阿帕期间或之后是否发生变化，将在用修饰的核苷酸标记新转录的mRNA之后立即或之后很久评估来自质粒的HTT同种型的长度。这些研究是具体目标1的重点。突变HTT同种型长度的变化可能影响下游加工，因为3'UTR含有许多指导mRNA稳定性和定位的基序。为了测试这一点，将通过转录停滞和qPCR（衰变）或荧光原位杂交（定位）在正常和疾病细胞中测定突变型和野生型HTT同种型的衰变和定位。为了验证差异是由于同种型长度引起的，将在用单一HTT同种型转染的细胞模型中测定同种型定位和衰变。这些研究是具体目标2的重点。总之，这些研究将阐明突变体和野生型mRNA阿帕，衰变和定位的差异。然后可以设计等位基因特异性小RNA来靶向突变亚型特有的mRNA区域或细胞区室，从而改善对这种毁灭性疾病的治疗。