Functional consequences of mutations in spliceosomal small nuclear RNAs

项目摘要 从前信使核糖核酸转录本中去除内含子是几乎所有人类表达的必要步骤 基因。这个项目的目标是确定分子、细胞和组织特异性的后果 U4atac单链RNA基因(RNU4ATAC)突变是罕见和严重人类 发育障碍小头骨发育不良I型原始侏儒症(MOPD I) 综合征(RS)和Lowry-Wood综合征(LWS)。次要类的剪接需要U4atac SnRNA 依赖于U12的内含子。含有这些内含子的基因在MOPD I和RS患者细胞中剪接得很差。我们 假设在MOPD I、RS和LWS中看到的各种病理是由于剪接减少或错配- 少量包含U12依赖内含子的基因的剪接。此外，我们发现， RNU4ATAC基因和其他SNRNA基因一样，含有大量的低频单核苷酸 在所有人类群体中都存在多态，为这些隐性疾病提供了突变来源。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，以 调查由这些突变引起的剪接中的特定缺陷(S)。1)我们将使用这些细胞系来分析 使用高通量方法研究所有可能的U4atac单链RNA突变对细胞生长的影响。基于 这些结果和在不同证候患者身上观察到的突变情况，我们将产生细胞系 对各种突变进行纯合，并使用生化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的特点是小头畸形和无脑畸形，我们使用MOPD I 患者来源的人iPS细胞产生并研究脑内有机物质。我们还产生了 小鼠突变的U4atac模型，概括了许多MOPD I病理。他们还会患上严重的糖尿病 两个月大的时候。随着疾病的发展，我们将研究小鼠胰腺的细胞表型 描述发育障碍的特征。3)我们将使用rna-seq和其他人类细胞的分子分析。 和小鼠组织将特定的病理与靶基因剪接缺陷联系起来。这些目标将是 通过正确拼接的基因表达恢复了基因的表达。成功完成这些任务 研究将促进我们对剪接体剪接的理解，确定人类 并确定U12依赖的剪接在基因表达和发育中的作用。

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.