Functional characterization of the Bronx waltzer deafness gene

布朗克斯华尔兹耳聋基因的功能特征

基本信息

批准号：
8494605
负责人：
Botond Banfi
金额：
$ 30.48万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
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/8494605
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

项目摘要

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）：具有耳聋的突变小鼠的表征导致了听力研究的重要发现，但是在一些公开可用的小鼠菌株中引起聋哑的基因缺陷仍然无法识别。这些菌株之一是Bronx Waltzer（BV），该菌株在1979年在纯合突变体中观察到的听力损失和“ Waltzing”行为。对BV小鼠的组织学分析显示，在围产期期间，无需伴随外毛细胞的损害，在围产期内有选择性丧失人工耳蜗内毛细胞和前庭毛细胞。我们的初步研究确定了BV小鼠菌株中引起聋的基因缺陷。该应用的目的是在功能上表征BV小鼠的耳聋基因。我们的中心假设是，BV突变截断了新型的剪接蛋白SER/ARG-RE-RE-RE-RE-RE-RE-RE-RE-RE-RE-RE-RE-RECTIVE矩阵4（SRRM4），以至于该蛋白无法介导关键的RNA剪接事件，这是内毛细胞和前庭毛细胞存活所需的必要所需的。该假设基于我们的初步研究，该研究：1）确定了BV小鼠的SRRM4基因中的缺失，2）用SRRM4的srRM4拯救了BV表型，3）3）SRRM4在毛细胞和神经元中表达了SRRM4，4）揭示了几个与MIRN的替代性MRN的相关性，并在MRN的替代方面表明了MRN的替代性。 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.我们将通过执行3个特定目的来检验中心假设：1）确定BV缺损的发胶选择性的分子基础，2）描述SRRM4调节前MRNA剪接的分子机制，以及3）探索BV小鼠中毛孔损失的病因学。在第一个目标下，我们将使用敲除小鼠评估SRRM4和SRRM3之间残留的SRRM4功能或冗余的程度，以说明BV缺陷的细胞类型特异性。在第二个目标下，我们将使用微型烯，RNA - 蛋白交联，免疫沉淀和FRET来描述SRRM4调节替代剪接的分子机制。在第三个目标下，我们将在器官培养环境中使用病毒基因递送来确定哪些已识别的剪接缺陷是BV小鼠中观察到的发型细胞损失的基础。拟议的研究具有创新性，因为它介绍了一个新颖的概念，即听力需要共同调节的替代剪接事件网络，并且因为它采用了尖端技术（外显子微阵列和新的生物信息学工具）来测试中央假设。拟议的研究之所以重要，是因为调节耳蜗和前庭系统中必不可少的RNA剪接的第一个耳聋基因的鉴定和表征将导致发现新型分子机制，蛋白质和蛋白质同种型，这些机制和蛋白质同种型是毛发细胞分化和存活所必需的。