Functional characterization of the Bronx waltzer deafness gene

描述（由申请人提供）：耳聋突变小鼠的表征在听力研究中取得了重要发现，但在一些公开的小鼠品系中，导致耳聋的基因缺陷仍未确定。其中一种菌株是布朗克斯华尔兹（bv），它在1979年被描述为在纯合突变体中观察到的听力丧失和“华尔兹”行为。对bv小鼠的组织学分析显示，围产期耳蜗内毛细胞和前庭毛细胞选择性丧失，外毛细胞无损伤。我们的初步研究发现了bv小鼠品系中引起耳聋的基因缺陷。本应用程序的目的是对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Ser/ arg - repeat matrix 4 (SRRM4)，使得该蛋白无法介导内毛细胞和前庭毛细胞存活所必需的关键RNA剪接事件。这个假设是基于我们的初步研究，其中：1)发现了bv小鼠SRRM4基因缺失，2)用SRRM4转基因挽救了bv表型，3)发现SRRM4在毛细胞和神经元中表达，4)揭示了bv内耳中几种mrna的选择性剪接异常，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)揭示了SRRM4及其同源物SRRM3可能在某些细胞类型中具有冗余功能。7)发现了与SRRM4相互作用的必要剪接因子，8)发现了在内耳中异常剪接的前mrna共享一个核苷酸基序。我们将通过三个具体目标来验证我们的中心假设：1)确定毛细胞选择性bv缺陷的分子基础，2)描述SRRM4调节前mrna剪接的分子机制，以及3)探索bv小鼠毛细胞脱落的病因。在第一个目标下，我们将使用敲除小鼠来评估SRRM4功能的残留或SRRM4和SRRM3之间的冗余在多大程度上解释bv缺陷的细胞类型特异性。在第二个目标下，我们将使用迷你基因、rna -蛋白交联、免疫沉淀和FRET来描绘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在bv小鼠中观察到的剪接缺陷中哪些是导致毛细胞丢失的原因。这项研究的创新之处在于，它引入了一个新的概念，即听觉需要一个共同调节的可选剪接事件网络，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）来检验中心假设。这项研究具有重要意义，因为鉴定和表征耳蜗和前庭系统中调节必需RNA剪接的第一耳聋基因将导致发现毛细胞分化和生存所需的新分子机制、蛋白质和蛋白质异构体。

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.