Cellular impact of X-linked dyskeratosis congenita

X连锁先天性角化不良的细胞影响

• 批准号: 9545059
• 负责人: U THOMAS MEIER
• 金额: $ 6.82万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9545059
• 关键词: Address; Affect; Antibodies; Biogenesis; Biological; Blood Cells; CRISPR/Cas technology; Cell Line; Cell model; Cell physiology; Cells; Complement; Complex; Cryoelectron Microscopy; Crystallization; Down-Regulation; Dyskeratosis Congenita; Electron Microscopy; Fibrinogen; Human; Immunoprecipitation; Impairment; In Vitro; Infection; Inherited; Length; Life; Light; Link; Malignant Neoplasms; Maps; Mediating; Methods; Missense Mutation; Modification; Molecular; Molecular Chaperones; Mutation; NAP57; Negative Staining; Nuclear; Nuclear Structure; Orthologous Gene; Outcome; Oxygen; Patients; Prostate; Protein Biosynthesis; Proteins; RNA; RNA Splicing; RNA immunoprecipitation sequencing; Regulation; Ribonucleoproteins; Ribosomes; Role; Small Nuclear RNA; Small Nucleolar RNA; Small Nucleolar Ribonucleoproteins; Son; Structure; Techniques; Technology; Telomerase; Telomerase RNA Component; Telomere Maintenance; Therapeutic; Untranslated RNA; X-Linked Dyskeratosis Congenita; Yeasts; base; bone marrow failure syndrome; disease phenotype; established cell line; experimental study; fighting; improved; in vivo; insight; knock-down; mRNA Precursor; mutant; next generation sequencing; novel; overexpression; prevent; telomere; tool

PROJECT SUMMARY Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome. Mutations in NAP57 (aka dyskerin) account for about half of the genetically characterized cases of DC and constitute the severe X-linked form of DC (X-DC). NAP57 is the corner stone of all H/ACA ribonucleoproteins (RNPs), one of the two major classes of small nucleolar RNPs (snoRNPs). As such, NAP57 is essential for the stable expression of over 500 different H/ACA RNAs including human telomerase RNA (hTR). H/ACA RNPs are important for many basic cellular processes including ribosome biogenesis, regulation of protein synthesis, pre-mRNA splicing, and telomere maintenance. While it is well documented that DC impacts hTR and telomeres, it is controversial if and how X-DC mutations affect other H/ACA RNPs and their function. In unbiased fashion, this proposal examines the impact of DC on all H/ACA RNPs in three specific aims. First, we will characterize the H/ACA RNAome in general and in X-DC. We will exploit our specific antibodies against NAP57 to precipitate all associated H/ACA RNAs and identify and quantify them by next-generation sequencing (NAP57 RIPseq). Now, we will apply NAP57 RIPseq to determine the relative abundance of H/ACA RNAs in healthy carriers of X-DC and their affected sons. In addition to defining the complete cellular H/ACA RNA landscape, our studies will pinpoint the changes that occur in patient cells and underlie the disease phenotype. Second, we will define the role of Cajal bodies (CBs) in H/ACA RNP biogenesis and in X-DC. CBs are micron-sized nuclear bodies whose function, the maturation and modification of non-coding RNAs (e.g. hTR), is compromised in DC. We developed model cell lines using CRISPR/Cas 9 technology to knockdown Nopp140, a partner of NAP57, which results in the specific down regulation of NAP57 in CBs but not nucleoli, mimicking a deficiency observed in DC. We propose to analyze these cells using NAP57 RIPseq and common cell biological approaches to compare the results to those of DC patient cells. Third, we will visualize the structure of wild type and mutant NAP57 +/- SHQ1. Before association with an H/ACA RNA, NAP57 is complexed with the chaperone SHQ1. The over 50 DC mutations in NAP57 impair this interaction diminishing NAP57 in the cell. To visualize the molecular impact of DC mutations, we propose to build on our negative-stain electron microscopy (EM) of human full-length NAP57 to visualize by cryo-EM the structures of wild type and mutant NAP57 alone and in complex with SHQ1. In summary, our studies will inform on the molecular mechanism of DC and move the field forward by establishing the complete cellular H/ACA RNAome, shedding light on the function of CBs, and generating a first structure of full- length wild type and mutant NAP57. The latter will aid developing therapeutic approaches for DC, as well as for certain cancers, e.g. prostate, that are also characterized by mutations in NAP57 and SHQ1.

项目总结 先天性角化不良(DC)是一种遗传性骨髓衰竭综合征。NAP57基因突变 (又名dyskerin)约占DC遗传特征病例的一半，并构成 严重的X连锁DC(X-DC)。NAP57是所有H/ACA核糖核蛋白(RNP)的基石， 两大类小核仁RNP(SnoRNPs)之一。因此，NAP57对于 稳定表达500多种不同的H/ACA RNA，包括人端粒酶RNA(HTR)。H/ACA RNPs在许多基本的细胞过程中都是重要的，包括核糖体的生物发生，调节 蛋白质合成、前信使核糖核酸剪接和端粒维持。虽然有很好的记录表明DC 影响hTR和端粒，X-DC突变是否以及如何影响其他H/ACA RNP是有争议的 以及它们的功能。该提案以不偏不倚的方式研究了DC对所有H/ACA RNP的影响 三个具体目标。首先，我们将描述H/ACA RNAome的一般特征和X-DC中的特征。我们会 利用我们针对NAP57的特异性抗体沉淀所有相关的H/ACA RNA并鉴定 并通过下一代测序(NAP57 RIPseq)对它们进行量化。现在，我们将应用NAP57 RIPseq 健康X-DC携带者及其受累人群H/ACA RNA相对丰度的测定 儿子们。除了定义完整的细胞H/ACA RNA图景外，我们的研究还将精确定位 在患者细胞中发生的变化，是疾病表型的基础。第二，我们将明确角色 在H/ACA RNP生物发生和X-DC中的Cajal小体(CBS)。CBS是微米大小的核天体 其功能，即非编码RNA的成熟和修饰(例如HTR)，在DC中受到损害。 我们使用CRISPR/Cas 9技术开发了模型细胞系，以击倒Nopp140，Nopp140是 NAP57，导致CBS中NAP57的特异性下调，但不是核仁，模拟 在DC观察到缺乏症。我们建议使用NAP57 RIPseq和普通细胞来分析这些细胞 用生物学方法将结果与DC患者细胞的结果进行比较。第三，我们将可视化 野生型和突变型NAP57+/-SHQ1的结构。在与H/ACA RNA结合之前，NAP57是 与伴侣SHQ1络合。NAP57中超过50个DC突变会削弱这种相互作用 细胞内的NAP57逐渐减少。为了可视化DC突变的分子影响，我们建议建立 在我们的人全长NAP57的负染电子显微镜(EM)上用冷冻-EM显示 野生型和突变型NAP57单独以及与SHQ1复合的结构。总而言之，我们的研究 将揭示DC的分子机制，并通过建立完整的 细胞H/ACA RNAome，揭示CBS的功能，并产生完整的第一结构- 长度野生型和突变型NAP57。后者将帮助开发DC的治疗方法，因为 以及某些癌症，例如前列腺癌，其特征也是NAP57和SHQ1突变。