Non-redundant functions of Hu proteins as neuron-specific splicing regulators

Hu蛋白作为神经元特异性剪接调节剂的非冗余功能

• 批准号: 8206867
• 负责人: Melissa N Hinman
• 金额: $ 0.58万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8206867
• 关键词: Accounting; Affect; Alternative Splicing; Antigen Targeting; Biochemical; Biological Assay; Biological Process; Cell Count; Cell Line; Cytoplasmic Granules; Data; Disease; Event; Evolution; Exhibits; Exons; Family; Family member; Genes; Glutamates; In Vitro; Lead; Learning; Light; Measures; Melissa; Memory; Methods; Modeling; Molecular; Mus; Mutation; NF1 gene; Nervous system structure; Neurofibromatoses; Neurofibromatosis 1; Neurologic; Neurons; Nuclear Extract; Output; Paraneoplastic Syndromes; Population; Preparation; Process; Protein Family; Proteins; Protocols documentation; RNA Interference; RNA Splicing; Regulation; Reporter; Research; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology; Severities; Spliceosome Assembly Pathway; Syndrome; System; Time; Tissues; Vertebrates; cell type; embryonic stem cell; immortalized cell; knock-down; mRNA Expression; mRNA Precursor; member; neuron development; novel; overexpression; public health relevance; research study; tool

DESCRIPTION (provided by applicant): I am investigating the hypothesis that Hu protein family members have non-redundant functions as negative regulators of the alternative splicing of neurofibromatosis type I (NF1) exon 23a. This research will help to determine the mechanisms behind how these splicing suppressors have diverged in function, and will also shed light on the mechanisms by which Hu proteins regulate splicing, which is not well understood. Preliminary data indicate that HuC is a strong suppressor and HuB a weak suppressor of NF1 exon 23a inclusion. In my project, I will investigate whether HuB and HuC vary in ability to regulate endogenous NF1 splicing in primary mouse cerebellar granule neurons. In addition, I will investigate the mechanism of the difference between HuB and HuC in splicing suppressor potency by performing in vitro splicing and spliceosome assembly assays. I will perform these assays using a novel system involving preparation of nuclear extract from mouse embryonic stem cells differentiated into neurons rather than from immortalized cell lines or whole tissues containing a heterogeneous mixture of cell types. Public Health Relevance: Alternative splicing is a process that is critical for many functions within the nervous system, such as neuronal development, and errors in alternative splicing can lead to disease, making it important to understand how the process is regulated. My study will help to elucidate the mechanism by which the Hu protein family regulates neurofibromatosis type I (NF1) exon 23a inclusion, a splicing event that is important for NF1 function and could possibly affect severity of NF1 disease. In addition, my studies will increase understanding of the molecular functions of the Hu proteins, which are associated with a disease called paraneoplastic syndrome.

描述(申请人提供)：我正在研究一种假设，即HU蛋白家族成员作为I型神经纤维瘤病(NF1)外显子23a选择性剪接的负调控因子具有非冗余功能。这项研究将有助于确定这些剪接抑制因子在功能上的差异背后的机制，也将有助于阐明HU蛋白调控剪接的机制，这一机制尚不清楚。初步数据表明，Huc是NF1外显子23a包涵体的强抑制因子，Hub是弱抑制因子。在我的项目中，我将研究Hub和Huc在调节原代小脑颗粒神经元内源性NF1剪接方面是否存在差异。此外，我还将通过体外剪接和剪接体组装实验来研究Hub和Huc在剪接抑制活性方面存在差异的机制。我将使用一种新的系统进行这些分析，该系统涉及从分化为神经元的小鼠胚胎干细胞制备核提取液，而不是从永生化细胞系或包含不同类型细胞混合的整个组织中制备核提取液。与公共卫生相关：选择性剪接是一个对神经系统内的许多功能至关重要的过程，例如神经元发育，而选择性剪接中的错误可能导致疾病，因此了解这一过程是如何调控的很重要。本研究将有助于阐明HU蛋白家族调控神经纤维瘤病I型(NF1)外显子23a包涵体的机制，这是一个对NF1功能至关重要的剪接事件，可能会影响NF1疾病的严重程度。此外，我的研究将增加对Hu蛋白分子功能的理解，这些蛋白与一种名为副肿瘤综合征的疾病有关。