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

 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。尽管研究人员正在开发小干扰 RNA 来靶向和降解突变 mRNA，但尚无有效的治疗或治愈方法。由于正常的 HTT 蛋白对于神经元的存活至关重要，因此治疗的目的是避免野生型 mRNA。如果突变型和野生型等位基因在转录后经过不同的处理，则可能特异性靶向突变型 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。一种可能性是突变体 mRNA 被加工成具有不同定位和稳定性的不同亚型。突变型和野生型 HTT mRNA 以两种 3'UTR 亚型存在。事实上，初步数据表明突变型 mRNA 比野生型 mRNA 更有可能以较长的亚型存在。拟议的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。由于该突变邻近参与选择性多聚腺苷酸化 (APA) 的基因区域，因此扩增可能会破坏突变体 HTT APA 并导致向更长亚型的转变。如果是这样，其他 mRNA 和其他等位基因应该正常处理。为了确定异构体长度的变化是否是 HTT 特异性的，将通过 qPCR 和深度测序评估杂合人类样本中 HTT 和其他 APA mRNA 的长度。为了验证扩增是否有效，将通过 qPCR 测量有或没有扩增的转染质粒的同种型长度。为了评估 HTT 同工型长度在 APA 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 之后立即或很久之后评估来自质粒的 HTT 同工型长度。这些研究是特定目标 1 的重点。突变体 HTT 同工型长度的变化可能会影响下游加工，因为 3'UTR 包含许多指导 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分析突变型和野生型 HTT 亚型的衰变和定位。为了验证差异是由于同种型长度造成的，将在用单一 HTT 同种型转染的细胞模型中分析同种型定位和衰减。这些研究是特定目标 2 的重点。这些研究将共同​​阐明突变型和野生型 mRNA APA、衰变和定位的差异。然后，可以设计等位基因特异性小 RNA，以靶向突变亚型特有的 mRNA 区域或细胞区室，从而改善这种破坏性疾病的治疗。

 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.