Functional characterization of the Bronx waltzer deafness gene

描述(由申请人提供)：突变型耳聋小鼠的特征在听力研究中取得了重要发现，但少数公开提供的小鼠品系中导致耳聋的基因缺陷仍未确定。其中一种是Bronx Waltzer(BV)，它于1979年被描述为在纯合突变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，而不伴随外毛细胞的损害。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。这项应用的目的是从功能上表征BV小鼠的耳聋基因。我们的中心假设是，BV突变截断了新的剪接体蛋白Ser/Arg重复基质4(SRRM4)，使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。这一假设是基于我们的初步研究：1)发现BV小鼠SRRM4基因的缺失，2)用SRRM4转基因挽救BV的表型，3)表明SRRM4在毛细胞和神经元中表达，4)发现BV内耳中几个mRNAs的选择性剪接异常，并且受影响的mRNAs编码功能相关的基因，5)表明剪接缺陷是毛细胞特有的，6)揭示SRRM4及其同源物SRRM3可能在某些细胞类型中具有冗余功能，7)发现与SRRM4相互作用的基本剪接因子，8)表明在BV内耳中异常剪接的前mRNAs共享一个核苷酸基序。我们将通过实现三个具体目标来验证我们的中心假设：1)确定BV缺陷毛细胞选择性的分子基础，2)描述SRRM4调节Pre-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SRRM4的残留功能或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Miniges、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丢失的基础。这项拟议的研究具有创新性，因为它引入了一个新的概念，即听力需要一个共同调节的选择性剪接事件网络，而且它使用了尖端技术(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。这项拟议的研究具有重要意义，因为第一个耳聋基因的鉴定和鉴定将导致发现毛细胞分化和生存所需的新的分子机制、蛋白质和蛋白质亚型。

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.