Functional consequences of mutations in spliceosomal small nuclear RNAs

剪接体小核RNA突变的功能后果

• 批准号: 10221000
• 负责人: RICHARD A PADGETT
• 金额: $ 47.1万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10221000
• 关键词: Affect; Age-Months; Alleles; Architecture; Bar Codes; Biochemical; Biochemistry; Biological Assay; Brain; Candidate Disease Gene; Cell Line; Cell Survival; Cells; Cerebrum; Characteristics; Competence; Complementary DNA; Defect; Development; Diabetes Mellitus; Disease; Embryo; Engineering; Excision; Frequencies; Gene Expression; General Population; Genes; Genetic Polymorphism; Genomics; Genotype; Goals; Growth; Health; Human; Human Biology; Immune System Diseases; Introns; Knowledge; Light; Link; Lowry-Wood syndrome; Mammals; Mental deficiency; Methods; Microcephaly; Migration Assay; Minor; Modeling; Molecular; Molecular Analysis; Mus; Mutant Strains Mice; Mutation; Neurons; Organ; Organoids; Pancreas; Pathogenesis; Pathogenicity; Pathology; Patients; Pattern; Phenotype; Point Mutation; Population; RNA Primary Transcript; RNA Splicing; Role; Single Nucleotide Polymorphism; Small Nuclear RNA; Spliceosomes; Suggestion; Surface; Syndrome; System; Techniques; Testing; Tissue-Specific Splicing; Tissues; Transcript; Transgenes; Untranslated RNA; Variant; base; cell growth; cell type; developmental disease; disease-causing mutation; human disease; human migration; human model; improved; in vivo; induced pluripotent stem cell; lissencephaly; mRNA Precursor; migration; mouse model; mutant; osteodysplastic primordial dwarfism; pancreas development; pup; restoration; skeletal abnormality; tool; transcriptome sequencing

Project Summary The removal of introns from pre-mRNA transcripts is an essential step in the expression of almost all human genes. The goal of this project is to determine the molecular, cellular and tissue-specific consequences of mutations in the U4atac snRNA gene (RNU4ATAC) that are the cause of the rare and severe human developmental disorders microcephalic osteodysplastic primordial dwarfism type I (MOPD I), Roifman Syndrome (RS) and Lowry-Wood Syndrome (LWS). U4atac snRNA is required for the splicing of minor class U12- dependent introns. Genes containing these introns are poorly spliced in MOPD I and RS patient cells. We hypothesize that the various pathologies seen in MOPD I, RS and LWS are caused by reduced splicing or mis- splicing of a small number of genes containing U12-dependent introns. In addition, we have found that the RNU4ATAC gene, as well as other snRNA genes, harbor large numbers of low frequency single nucleotide polymorphisms in all human populations, supplying a mutational origin for these recessive disorders. We have developed cell lines, including patient-derived and gene-edited human iPS cells, and new assay systems to investigate the specific defect(s) in splicing caused by the mutations. 1) We will use these cell lines to analyze the effects of all possible U4atac snRNA mutations on cell growth using a high-throughput method. Based on these results and the mutations observed in patients with the different syndromes, we will generate cell lines homozygous for various mutations and employ biochemical techniques to characterize their functional defects. 2) We will determine the differentiation potential of human iPS cells with a variety of disease-linked mutations. For example, since severe MOPD I is characterized by microcephaly and lissencephaly, we are using MOPD I patient-derived human iPS cells to generate and study cerebral brain organoids. We have also generated mouse mutant U4atac models that recapitulate many MOPD I pathologies. They also develop severe diabetes by two months of age. We will study the cellular phenotypes of the mouse pancreas as the disease develops to characterize the developmental disorder. 3) We will use RNA-seq and other molecular analyses of human cells and mouse tissues to relate specific pathologies to defects in splicing of target genes. These targets will be validated through restoration of gene expression by properly spliced cDNA. Successful completion of these studies will advance our understanding of spliceosomal splicing, define the molecular causes of human diseases and determine the role of U12-dependent splicing in gene expression and development.

项目摘要 前体mRNA转录物中内含子的去除是几乎所有人类基因表达的重要步骤。 基因.该项目的目标是确定的分子，细胞和组织特异性的后果， U4 atac snRNA基因（RNU 4ATAC）的突变是导致罕见和严重的人类 发育障碍性小头骨发育不良性原始侏儒症I型，罗夫曼 综合征（RS）和Lowry-Wood综合征（LWS）。U4 atac snRNA是剪接小类 U12依赖性内含子。含有这些内含子的基因在MOPD I和RS患者细胞中剪接不良。我们 假设在MOPD I、RS和LWS中观察到的各种病理是由剪接减少或错误剪接引起的。 含有U12依赖性内含子的少量基因的剪接。此外，我们还发现， RNU 4ATAC基因与其他snRNA基因一样，含有大量的低频单核苷酸， 在所有人群中的多态性，提供了这些隐性疾病的突变起源。我们有 开发的细胞系，包括患者来源的和基因编辑的人类iPS细胞，以及新的检测系统， 研究由突变引起的剪接中的特定缺陷。1)我们将用这些细胞系来分析 使用高通量方法研究所有可能的U4 atac snRNA突变对细胞生长的影响。基于 根据这些结果和在不同综合征患者中观察到的突变，我们将产生细胞系， 它们是各种突变的纯合子，并采用生物化学技术来表征其功能缺陷。 2)我们将确定具有各种疾病相关突变的人iPS细胞的分化潜力。 例如，由于重度MOPD I的特征是小头畸形和无脑畸形， 患者来源的人iPS细胞来产生和研究大脑类器官。我们还产生了 小鼠突变体U4 atac模型，重现了许多MOPD I病理。他们还会患上严重的糖尿病 两个月大的时候。我们将研究小鼠胰腺的细胞表型， 描述发育障碍。3)我们将使用RNA-seq和其他人类细胞的分子分析 和小鼠组织来将特定的病理与靶基因剪接中的缺陷相关联。这些目标将是 通过正确剪接的cDNA恢复基因表达来验证。成功完成这些 研究将促进我们对剪接体剪接的理解，确定人类疾病的分子原因， 疾病和确定U12依赖性剪接在基因表达和发育中的作用。