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: 8185723
• 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/8185723
• 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. PUBLIC HEALTH RELEVANCE: The proposed project is relevant to public health in that it is expected to characterize a novel deafness gene, and to thereby advance our knowledge in the field of audiology. The resulting improvement in our understanding of the pathophysiology of the hearing organ is expected to lead to improved molecular diagnosis of hearing impairment, as well as to improved clinical care for patients with hearing loss.

描述（由申请人提供）：耳聋突变小鼠的特征描述导致了听力研究中的重要发现，但在一些公开可用的小鼠品系中导致耳聋的基因缺陷仍未确定。这些菌株之一是布朗克斯华尔兹（bv），这是在1979年描述的听力损失和“华尔兹”行为观察纯合子突变。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有选择性丢失，而外毛细胞没有伴随损伤。我们的初步研究确定了bv小鼠品系中导致肥胖的基因缺陷。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突变截断了新的剪接体蛋白丝氨酸/精氨酸重复矩阵4（SRRM 4）的方式，这种蛋白质无法介导的关键RNA剪接事件所必需的内毛细胞和前庭毛细胞的生存。这一假设是基于我们的初步研究，其中：1）鉴定了bv小鼠SRRM 4基因的缺失，2）用SRRM 4转基因挽救了bv表型，3）显示SRRM 4在毛细胞和神经元中表达，4）揭示了几种mRNA的选择性剪接在bv内耳中是异常的，并且受影响的mRNA编码功能相关的基因，5）显示剪接缺陷是毛细胞特异性的，6）揭示SRRM 4及其同源物SRRM 3可能在某些细胞类型中具有冗余功能，7）鉴定与SRRM 4相互作用的必需剪接因子，和8）显示在bv内耳中异常剪接的前mRNA共享核苷酸基序。我们将通过3个具体目标来检验我们的中心假设：1）确定bv缺陷毛细胞选择性的分子基础，2）描绘SRRM 4调节前mRNA剪接的分子机制，3）探索bv小鼠毛细胞丢失的病因。在第一个目标下，我们将使用基因敲除小鼠来评估SRRM 4和SRRM 3之间的残余SRRM 4功能或冗余度对bv缺陷的细胞类型特异性的影响程度。根据第二个目标，我们将使用小基因，RNA-蛋白质交联，免疫沉淀和FRET描绘SRRM 4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，以确定在bv小鼠中观察到的毛细胞损失的基础是哪些已识别的剪接缺陷。拟议的研究是创新的，因为它引入了新的概念，即听力需要一个共同调节的选择性剪接事件网络，并且因为它采用了尖端技术（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。这项研究意义重大，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发现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。 公共卫生关系：拟议的项目是相关的公共卫生，因为它有望表征一种新的耳聋基因，从而提高我们在听力学领域的知识。由此产生的听力器官的病理生理学的理解的改善，预计将导致改善听力障碍的分子诊断，以及改善听力损失患者的临床护理。