Alternative splicing in the complex Drosophila dumpy gene

复杂果蝇矮胖基因中的选择性剪接

• 批准号: 7923972
• 负责人: ROSS Joseph MACINTYRE
• 金额: $ 19.25万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7923972
• 关键词: Adhesions; Affect; Alternative Splicing; Animal Model; Apical; Apoptosis; Cells; Collection; Complement; Complex; Defect; Development; Drosophila genus; Drosophila melanogaster; Endoglin; Epithelial Cells; Event; Exons; Extracellular Matrix; Extracellular Matrix Proteins; Fertility; Genes; Human; In Situ Hybridization; Investigation; Lead; Light; Mutation; Neuraxis; Nonsense Mutation; Nucleotides; Pattern; Positioning Attribute; Process; Protein Isoforms; RNA Splicing; Recombinants; Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Transduction; Site; Staging; Structure; Surface; Tertiary Protein Structure; Tissues; Trans-Splicing; Transcript; Vascular Diseases; Zona Pellucida; hearing impairment; human disease; mutant; protein complex; public health relevance

DESCRIPTION (provided by applicant): In the central nervous system, protein isoform diversity is responsible, at least in part, for its complexity and its many orchestrated activities. This proposal concerns the role of alternative splicing in the differentiation and function of various extracellular matrices (ECMs) of epithelial cells during the development of the model organism, Drosophila melanogaster. We will explore the possibility that different ECM protein isoforms, generated by both cis and trans alternative splicing of mRNAs encoded by the complex dumpy gene, characterize ECMs at the apical surfaces of epithelial cells in different tissues at different developmental stages. Subsequent investigations will determine if the isoforms resulting from alternative splicing are involved in various ECM functions such as adhesion, apoptosis, remodeling and signaling. Since Zona pellucida (ZP) domain proteins, like Dumpy, are important components of vertebrate ECMs, these studies will directly relate to certain human defects. Thus, mutations of the ZP domain protein, alpha-tectorin, lead to hearing loss and mutations in Endoglin are associated with a hemorrhagic vascular disease. Other ZP domain containing protein complexes are essential for fertility. An understanding of the structure and function of apical ECMs will shed light on these and other human diseases. Our approach will utilize a large collection of molecularly defined dumpy mutants which affect all or only a subset of Drosophila tissues and developmental stages. We will use primers located at strategic positions in the huge and complex dumpy gene for RT-PCR to detect cis alternative splicing events. Trans-splicing events will be examined in exons marked by complementing dumpy lethal nonsense mutations. Both the mutant sites themselves and single nucleotide differences (SNPs) will be used to detect recombinant transcripts. PUBLIC HEALTH RELEVANCE: Cells are held together in different tissues by means of the extracellular matrix (ECM). The ECM must be remodeled in the morphogenetic processes of development. We propose that, like the central nervous system, alternative splicing of ECM transcripts underlies its complexity, and will use the Drosophila dumpy gene to document this phenomenon.

描述（由申请人提供）：在中枢神经系统中，蛋白质同工型的多样性至少部分是由于其复杂性和许多精心策划的活动。该建议涉及替代剪接在模型生物体果蝇果蝇（Drosophila Melanogaster）开发过程中上皮细胞各种细胞外基质（ECM）的分化和功能中的作用。我们将探讨通过复合腐败基因编码的MRNA和反式替代剪接产生的不同ECM蛋白同工型的可能性，它表征了不同发育阶段不同组织上皮细胞的顶部表面的ECMS。随后的研究将确定替代剪接产生的同工型是否参与了各种ECM功能，例如粘附，凋亡，重塑和信号传导。由于Zona pellucida（ZP）结构蛋白（如Dumby）是脊椎动物ECM的重要组成部分，因此这些研究将直接与某些人类缺陷有关。因此，ZP结构蛋白，α-核蛋白的突变导致听力丧失和内尾突变与出血性血管疾病有关。其他含有蛋白质复合物的ZP结构域对于生育力至关重要。对顶端ECM的结构和功能的理解将阐明这些和其他人类疾病。我们的方法将利用大量分子定义的矮小突变体，这些突变体影响果蝇组织的全部或仅一部分和发育阶段。我们将使用位于巨大且复杂的矮小基因中的战略位置的引物来检测CIS替代剪接事件。通过补充谦卑的致命胡说突变，将在外显子中检查变形事件。突变位点本身和单核苷酸差异（SNP）将用于检测重组转录本。 公共卫生相关性：通过细胞外基质（ECM）将细胞固定在不同组织中。必须在发育的形态发生过程中重塑ECM。我们建议，像中枢神经系统一样，ECM转录本的替代剪接是其复杂性的基础，并将使用果蝇腐败基因记录这种现象。