The Role of Alternative Splicing Factor Sfrs10 in Neural Development

选择性剪接因子 Sfrs10 在神经发育中的作用

• 批准号: 7788630
• 负责人: RAHUL N KANADIA
• 金额: $ 9万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-02-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7788630
• 关键词: Affect; Alternative Splicing; Alzheimer' s Disease; Antibodies; Architecture; Autistic Disorder; Bioinformatics; Biological Assay; Biological Models; Birth Order; Brain; Cell Separation; Cells; Central Nervous System Part; Code; Complementary DNA; Complex; DNA; Data; Defect; Development; Disease; Drosophila genus; Electroporation; Embryo; Event; Exclusion; Exons; Fluorescence; Genes; Goals; House mice; Human; Human Genome; In Vitro; Infection; Knockout Mice; Lead; Link; Mammals; Masks; Messenger RNA; Motor Neurons; Mus; Mutation; Myotonic Dystrophy; Neural Retina; Neuraxis; Neuroglia; Neurons; Nonsense Codon; Northern Blotting; Pathogenesis; Pathway interactions; Pattern; Play; Process; Protein Isoforms; Proteins; Proteome; RNA; RNA Interference; RNA Splicing; Reporting; Research; Research Proposals; Resolution; Retina; Retinal; Reverse Transcriptase Polymerase Chain Reaction; Role; Set protein; Spinal Muscular Atrophy; Structure; Techniques; Technology; Tissues; Tretinoin; Ultrasonography; Vertebrates; Virus; Work; base; cell type; design; gain of function; ganglion cell; glutamate receptor type B; human fetus tissue; in vivo; injured; insight; interest; loss of function; nervous system development; neurodevelopment; numb protein; postnatal; public health relevance; relating to nervous system; research study; retinal progenitor cell; sex determination; tool; Affect; Alternative Splicing; Alzheimer' s Disease; Antibodies; Architecture; Autistic Disorder; Bioinformatics; Biological Assay; Biological Models; Birth Order; Brain; Cell Separation; Cells; Central Nervous System Part; Code; Complementary DNA; Complex; DNA; Data; Defect; Development; Disease; Drosophila genus; Electroporation; Embryo; Event; Exclusion; Exons; Fluorescence; Genes; Goals; House mice; Human; Human Genome; In Vitro; Infection; Knockout Mice; Lead; Link; Mammals; Masks; Messenger RNA; Motor Neurons; Mus; Mutation; Myotonic Dystrophy; Neural Retina; Neuraxis; Neuroglia; Neurons; Nonsense Codon; Northern Blotting; Pathogenesis; Pathway interactions; Pattern; Play; Process; Protein Isoforms; Proteins; Proteome; RNA; RNA Interference; RNA Splicing; Reporting; Research; Research Proposals; Resolution; Retina; Retinal; Reverse Transcriptase Polymerase Chain Reaction; Role; Set protein; Spinal Muscular Atrophy; Structure; Techniques; Technology; Tissues; Tretinoin; Ultrasonography; Vertebrates; Virus; Work; base; cell type; design; gain of function; ganglion cell; glutamate receptor type B; human fetus tissue; in vivo; injured; insight; interest; loss of function; nervous system development; neurodevelopment; numb protein; postnatal; public health relevance; relating to nervous system; research study; retinal progenitor cell; sex determination; tool

DESCRIPTION (provided by applicant): Through alternative splicing a single gene can generate functionally diverse set of proteins. Indeed, AS is invoked to reconcile the difference between the relatively few (~24,000) protein coding genes in the human genome to its vast proteome. Interestingly, of all the mammal tissues, the central nervous system (CNS) has the highest degree of AS. Thus, it is generally accepted that AS plays a crucial role in neural development, but there is a paucity of information on this issue. Given the complexity of CNS development combined with that of AS, we have chosen to employ the neural retina as our model system. The retina is derived from the CNS, is a relatively simple tissue, has a well defined laminar structure, it has six neural cell types and one glia, birth order of each cell type is known, and importantly it is the most accessible part of the CNS. The overall goal of this research proposal is to investigated the contribution of AS in neural development by studying an AS factor, Sfrs10 in the mouse retina. Previous studies in Drosophila have shown that Sfrs10 is required during sex determination and others have shown that Sfrs10 regulates the AS some neural genes. Based on this and our preliminary work the hypothesis underlying this proposal is that Sfrs10 is essential for neural cell fate determination and differentiation. There are two specific aims and the first specific aim will accomplish the following goals. We will characterize the expression pattern and AS of Sfrs10 during embryonic and postnatal retinal development. In addition, loss (RNAi) and gain of function will be performed by either in vivo DNA electroporation of a postnatal day 0 mouse retina or by in vitro electroporation of embryonic retinal explant cultures. We will employ ultrasound guided delivery technology to deliver viruses with either a gain or a loss of function construct into E10.5 embryo. A significant effort will be dedicated towards finding the targets of Sfrs10. Moreover, a conditional knockout mouse will be generated to validate the aforementioned experiments and provide a tool for the second specific aim. The second specific aim will investigate the AS status of the targets of Sfrs10 at the single cell resolution. The premise here is that tissues consists of several cell types and if each cell type splices an exon differently, then the use of entire tissue for analysis might mask this interesting fact. PUBLIC HEALTH RELEVANCE: Understanding the role of alternative splicing in development is essential to our understanding the underlying mechanism that leads to diseases. There are several diseases such as Alzheimer's disease, myotonic dystrophy, spinal muscular atrophy and autism that are linked to defects in alternative splicing of specific genes or are caused by mutations in genes that regulate the process of alternative splicing.

描述（由申请人提供）：通过替代剪接单个基因可以生成功能多样的蛋白质集。确实，被调用以调和人类基因组中相对较少的（约24,000）个蛋白质编码基因与其广泛蛋白质组之间的差异。有趣的是，在所有哺乳动物组织中，中枢神经系统（CNS）具有最高的AS。因此，人们普遍认为，由于在神经发育中起着至关重要的作用，但是关于这个问题的信息很少。鉴于中枢神经系统开发的复杂性与 因为，我们选择使用神经视网膜作为我们的模型系统。视网膜源自中枢神经系统，是一个相对简单的组织，具有良好的层状结构，它具有六种神经细胞类型和1个神经胶质，每种细胞类型的出生顺序是已知的，重要的是，它是CNS中最容易获得的部分。该研究建议的总体目标是通过研究小鼠视网膜中的AS SFRS10来研究AS在神经发育中的贡献。果蝇的先前研究表明，在性别确定过程中需要SFRS10，而其他研究表明SFRS10调节了某些神经基因。基于此和我们的初步工作，该提议的基本假设是SFRS10对于神经细胞命运的确定和分化至关重要。有两个具体的目标，第一个具体目标将实现以下目标。我们将在胚胎和产后视网膜发育过程中表征表达模式，如SFRS10。此外，通过体内DNA电穿孔的损失（RNAi）和功能增益将进行产后0小鼠视网膜的电穿孔或胚胎视网膜外植体培养物的体外电穿孔。我们将采用超声引导的输送技术，以增益或功能构建的损失传递到E10.5胚胎中。将为寻找SFRS10的目标做出的重大努力。此外，将生成条件敲除鼠标以验证上述实验，并为第二个特定目标提供工具。第二个特定目标将调查 SFRS10在单细胞分辨率下的靶标。这里的前提是组织由几种细胞类型组成，如果每种细胞类型都以不同的方式剪接外显子，则使用整个组织进行分析可能会掩盖这一有趣的事实。 公共卫生相关性：了解替代剪接在开发中的作用对于我们理解导致疾病的潜在机制至关重要。有几种疾病，例如阿尔茨海默氏病，肌营养不良症，脊柱肌肉萎缩和自闭症，与特定基因的替代剪接中的缺陷有关，或者是由调节替代旋转过程的基因突变引起的。