Functional characterization of the Bronx waltzer deafness gene

布朗克斯华尔兹耳聋基因的功能特征

• 批准号: 8681414
• 负责人: Botond Banfi
• 金额: $ 32.09万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-15 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8681414
• 关键词: Accounting; Affect; Alternative Splicing; Audiology; Behavior; Binding; Bioinformatics; Biological; Cell Death; Cell Differentiation process; Cell Survival; Cells; Cochlea; Complex; Conserved Sequence; Data; Defect; Development; Ear; Embryo; Equilibrium; Etiology; Event; Exons; Fluorescence Resonance Energy Transfer; Functional disorder; Gene Expression Profile; Genes; Goals; Hair Cells; Hearing; Homologous Gene; Hybrids; Immunoprecipitation; Inner Hair Cells; Knockout Mice; Knowledge; Labyrinth; Lead; Link; Mediating; Mediator of activation protein; Messenger RNA; Methods; Modeling; Molecular; Molecular Diagnosis; Mouse Strains; Mus; Mutagenesis; Mutant Strains Mice; Mutation; Neurons; Nucleotides; Organ; Organ Culture Techniques; Outer Hair Cells; Pathology; Patients; Perinatal; Phenotype; Protein Isoforms; Protein Overexpression; Proteins; Public Health; RNA; RNA Splicing; Recruitment Activity; Regulation; Research; Residual state; Sequence Analysis; Signal Transduction; Site; Specificity; System; Technology; Testing; Tissues; Transgenes; Vestibular Hair Cells; Viral; Work; Yeasts; base; cell type; clinical care; crosslink; deafness; dimer; disease-causing mutation; embryonic stem cell; gene function; hearing impairment; improved; in vivo; innovation; knock-down; mRNA Precursor; mutant; novel; protein crosslink; protein protein interaction; protein transport; research study; synaptogenesis; tool; viral gene delivery

DESCRIPTION (provided by applicant): The characterization of mutant mice with deafness has led to important discoveries in hearing research, yet the deafness-causing gene defects in a few publicly available mouse strains remain unidentified. One of these strains is Bronx waltzer (bv), which was described in 1979 with respect to the hearing loss and "waltzing" behavior observed in homozygous mutants. Histological analysis of bv mice revealed a selective loss of cochlear inner hair cells and vestibular hair cells during the perinatal period, without accompanying damage to outer hair cells. Our preliminary studies identified the deafness-causing gene defect in the bv mouse strain. The objective of this application is to functionally characterize the deafness gene of bv mice. Our central hypothesis is that the bv mutation truncates the novel spliceosomal protein Ser/Arg-repetitive matrix 4 (SRRM4) in such a way that this protein fails to mediate key RNA splicing events necessary for the survival of inner hair cells and vestibular hair cells. This hypothesis is based on our preliminary studies which: 1) identified a deletion in the SRRM4 gene of bv mice, 2) rescued the bv phenotype with an SRRM4 transgene, 3) showed that SRRM4 is expressed in hair cells and neurons, 4) revealed that alternative splicing of several mRNAs is aberrant in the bv inner ear and that the affected mRNAs encode functionally related genes, 5) showed that the splicing defects are hair-cell specific, 6) revealed that SRRM4 and its homolog SRRM3 may have redundant functions in certain cell types, 7) identified essential splicing factors that interact with SRRM4, and 8) showed that a nucleotide motif is shared by the pre-mRNAs that are aberrantly spliced in the bv inner ear. We will test our central hypothesis by carrying out 3 specific aims: 1) determine the molecular basis of the hair-cell selectivity of the bv defect, 2) delineate the molecular mechanism by which SRRM4 regulates pre-mRNA splicing, and 3) explore the etiology of hair-cell loss in the bv mice. Under the first aim, we will use knock-out mice to evaluate the extent to which residual SRRM4 function or redundancy between SRRM4 and SRRM3 accounts for the cell-type specificity of the bv defect. Under the second aim, we will use minigenes, RNA-protein crosslinking, immunoprecipitation, and FRET to delineate the molecular mechanism by which SRRM4 regulates alternative splicing. Under the third aim, we will use viral gene delivery in an organ culture setting to determine which of the identified splicing defects underlie the hair-cell loss observed in bv mice. The proposed research is innovative in that it introduces the novel concept that a jointly regulated network of alternative splicing events is required for hearing, and because it employs cutting-edge technologies (exon junction microarrays and new bioinformatics tools) to test the central hypothesis. The proposed study is significant because the identification and characterization of the first deafness gene that regulates essential RNA splicing in the cochlea and vestibular system will lead to the discovery of novel molecular mechanisms, proteins, and protein isoforms that are required for hair cell differentiation and survival.

描述（由申请人提供）：耳聋突变小鼠的表征在听力研究中带来了重要发现，但一些公开的小鼠品系中导致耳聋的基因缺陷仍未确定。其中一种菌株是 Bronx waltzer (bv)，它于 1979 年被描述为在纯合突变体中观察到的听力损失和“华尔兹”行为。对 bv 小鼠的组织学分析显示，在围产期，耳蜗内毛细胞和前庭毛细胞选择性丧失，但外毛细胞没有受到损害。我们的初步研究确定了 bv 小鼠品系中导致耳聋的基因缺陷。本申请的目的是对 bv 小鼠的耳聋基因进行功能表征。我们的中心假设是，bv 突变截短了新型剪接体蛋白 Ser/Arg 重复矩阵 4 (SRRM4)，使得该蛋白无法介导内毛细胞和前庭毛细胞生存所需的关键 RNA 剪接事件。这一假设基于我们的初步研究，其中：1）确定了 bv 小鼠 SRRM4 基因中的缺失，2）用 SRRM4 转基因挽救了 bv 表型，3）表明 SRRM4 在毛细胞和神经元中表达，4）揭示了 bv 内耳中几种 mRNA 的选择性剪接是异常的，并且受影响的 mRNA 编码功能相关 基因，5) 表明剪接缺陷是毛细胞特异性的，6) 表明 SRRM4 及其同源物 SRRM3 在某些细胞类型中可能具有冗余功能，7) 鉴定出与 SRRM4 相互作用的重要剪接因子，8) 表明 bv 内耳中异常剪接的前 mRNA 共享一个核苷酸基序。我们将通过实现 3 个具体目标来检验我们的中心假设：1）确定 bv 缺陷的毛细胞选择性的分子基础，2）描绘 SRRM4 调节前 mRNA 剪接的分子机制，3）探索 bv 小鼠毛细胞损失的病因学。在第一个目标下，我们将使用基因敲除小鼠来评估残余 SRRM4 功能或 SRRM4 和 SRRM3 之间的冗余在多大程度上解释了 bv 缺陷的细胞类型特异性。在第二个目标下，我们将使用小基因、RNA-蛋白质交联、免疫沉淀和 FRET 来描述 SRRM4 调节选择性剪接的分子机制。在第三个目标下，我们将在器官培养环境中使用病毒基因传递来确定哪些已识别的剪接缺陷是在 bv 小鼠中观察到的毛细胞损失的基础。拟议的研究具有创新性，因为它引入了听力需要共同调控的选择性剪接事件网络的新概念，并且因为它采用尖端技术（外显子连接微阵列和新的生物信息学工具）来测试中心假设。这项研究具有重要意义，因为第一个调节耳蜗和前庭系统中必需 RNA 剪接的耳聋基因的鉴定和表征将导致毛细胞分化和存活所需的新分子机制、蛋白质和蛋白质亚型的发现。