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: 9861050
• 负责人: U THOMAS MEIER
• 金额: $ 52.94万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 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 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9861050
• 关键词: Cellular; impact; linked; dyskeratosis; congenita

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.

项目摘要 先天性角化不良（DC）是一种遗传性骨髓衰竭综合征。NAP 57突变 (aka dyskerin）占DC遗传特征病例的一半左右，并构成了 严重X连锁型DC（X-DC）。NAP 57是所有H/ACA核糖核蛋白（RNP）的基石， 两大类小核仁RNP（snoRNP）之一。因此，NAP 57对于 稳定表达超过500种不同的H/ACA RNA，包括人端粒酶RNA（hTR）。H/ACA RNP对于许多基本的细胞过程是重要的，包括核糖体生物发生、细胞周期的调节、细胞周期的调控和细胞周期的调控。 蛋白质合成、前mRNA剪接和端粒维持。虽然有充分的证据表明，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 和它们的功能。在公正的方式，这项建议审查的影响，DC对所有H/ACA RNP在 三个具体目标。首先，我们将在一般和X-DC中表征H/ACA RNAome。我们将 利用我们针对NAP 57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（NAP 57 RIPseq）对其进行定量。现在，我们将应用NAP 57 RIPseq 确定健康X-DC携带者及其受影响者中H/ACA RNA的相对丰度， 个儿子除了定义完整的细胞H/ACA RNA景观外，我们的研究还将查明 患者细胞中发生的变化和疾病表型的基础。第二，我们要明确角色 Cajal小体（CB）在H/ACA RNP生物发生和X-DC中的表达。CB是微米大小的核体 其功能，非编码RNA（例如hTR）的成熟和修饰，在DC中受到损害。 我们使用CRISPR/Cas 9技术开发了模型细胞系，以敲除Nopp 140，Nopp 140是Nopp 140的伴侣。 NAP 57，其导致NAP 57在CB中而不是核仁中的特异性下调，模拟了NAP 57在CB中的表达。 在DC中观察到的缺陷。我们建议使用NAP 57 RIPseq和普通细胞分析这些细胞。 生物学方法来将结果与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NAP 57 +/-SHQ 1的结构。在与H/ACA RNA结合之前， 与伴侣SHQ 1复合。NAP 57中超过50个DC突变削弱了这种相互作用 减少细胞中的NAP 57。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NAP 57的负染色电子显微镜（EM）上，通过冷冻-EM观察 单独的野生型和突变型NAP 57以及与SHQ 1复合的结构。总之，我们的研究 将告知DC的分子机制，并通过建立完整的 细胞H/ACA RNAome，揭示CB的功能，并产生完整的第一个结构， 长度野生型和突变型NAP 57。后者将有助于开发DC的治疗方法， 以及对于某些癌症，例如前列腺癌，其特征也在于NAP 57和SHQ 1中的突变。