Role of rRNA Modifications in Dyskeratosis Congenita

rRNA 修饰在先天性角化不良中的作用

• 批准号: 7130793
• 负责人: Davide Ruggero
• 金额: $ 42.75万
• 依托单位: FOX CHASE CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7130793
• 关键词: bioinformatics; bioluminescence; cell cycle proteins; density gradient ultracentrifugation; enzyme inhibitors; gene mutation; genetic disorder; genetic regulatory element; genetic translation; genetically modified animals; high throughput technology; imaging /visualization /scanning; laboratory mouse; messenger RNA; nuclear proteins; polysomes; posttranslational modifications; protein biosynthesis; protein kinase; proteomics; reporter genes; ribosomal RNA; tumor suppressor genes; uracil nucleoside

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to elucidate the molecular and cellular basis for X-linked Dyskeratosis Congenita (X-DC), an inherited disease characterized by early mortality due to bone marrow failure as well as increased tumor susceptibility. The gene found mutated in X-DC, Dyskeratosis Congenita 1 (DKC1), is a pseudouridine synthase that mediates post-transcriptional modification of ribosomal RNA (rRNA) of both large and small ribosomal subunits through conversion of the uridine nucleotide (U) to pseudouridine (psi). The role of rRNA modifications in ribosome function and protein synthesis is poorly understood. We have generated DKC1 hypomorphic mice (DKC1m), which faithfully recapitulate all the clinical features of X-DC and are the first mouse model for severe aplastic anemia, a life threatening disease present in a heterogeneous group of disorders characterized by the failure in forming blood cells. DKC1m mice, also show increased cancer susceptibility and more than 50% of these animals develop carcinomas and B-cell lymphomas. We have shown that primary cells from DKC1m mice possess impairments in rRNA pseudouridylation and display decreased ribosome activity prior to disease onset. As outlined in the preliminary studies section, we have successfully utilized a proteomics strategy to identify a subset of mRNAs that are translationally impaired in DKC1m cells. These findings support a role for rRNA modifications in translation initiation of specific mRNAs, which harbor an internal ribosome entry site (IRES) positioned in their 5'UTR (5'untraslated region). The goals of the proposed study are to investigate the role of dyskerin and rRNA modification in control of protein synthesis. In addition, utilizing a molecular and genetic approach we will monitor translation regulation in vivo in DKC1m mice and genetically test the functional consequences of translational impairments in key target mRNAs towards the pathological features of X-DC. In Aim 1 we will expand on our proteomics strategy to characterize mRNAs, which rely on dyskerin activity and rRNA modifications for translation initiation. In Aim 2 we will define, in vivo, the role of defective IRES-dependent translation in X-DC pathogenesis utilizing a genetic approach as well as a novel live imaging system to follow IRES-dependent translation. Finally, in Aim 3 we will determine the molecular step(s) in which DKC1m ribosomes fail to initiate protein synthesis utilizing an in vitro reconstituted system. Lay abstract. Together these studies will build a deeper foundation for understanding the key genetic and molecular events that cause X-DC human disease and provide new animal models that will be vital for understanding the underlying causes of bone marrow failure and cancer susceptibility.

描述（由申请人提供）： 该提案的长期目标是阐明X连锁先天性角化不良（X-DC）的分子和细胞基础，X-DC是一种遗传性疾病，其特征在于由于骨髓衰竭以及肿瘤易感性增加而导致的早期死亡。在X-DC中发现突变的基因，先天性角化不良1（DKC 1），是一种假尿苷合酶，通过尿苷核苷酸（U）转化为假尿苷（psi）介导大小核糖体亚基的核糖体RNA（rRNA）的转录后修饰。rRNA修饰在核糖体功能和蛋白质合成中的作用知之甚少。我们已经产生了DKC 1 hypomorphic小鼠（DKC 1 m），它忠实地概括了X-DC的所有临床特征，并且是第一个严重再生障碍性贫血的小鼠模型，这是一种危及生命的疾病，存在于一组异质性疾病中，其特征在于无法形成血细胞。DKC 1 m小鼠也表现出癌症易感性增加，超过50%的这些动物发展为癌症和B细胞淋巴瘤。我们已经表明，DKC 1 m小鼠的原代细胞具有rRNA假尿苷酸化损伤，并在疾病发作前显示核糖体活性降低。如初步研究部分所述，我们成功地利用蛋白质组学策略鉴定了DKC 1 m细胞中功能受损的mRNA亚组。这些发现支持rRNA修饰在特定mRNA的翻译起始中的作用，所述特定mRNA在其5 'UTR（5'非翻译区）中具有内部核糖体进入位点（IRES）。本研究的目的是研究dyskerin和rRNA修饰在蛋白质合成控制中的作用。此外，利用分子和遗传学方法，我们将监测DKC 1 m小鼠体内的翻译调控，并对X-DC病理特征的关键靶mRNA的翻译损伤的功能后果进行遗传学测试。在目标1中，我们将扩展我们的蛋白质组学策略来表征mRNA，其依赖于dyskerin活性和翻译起始的rRNA修饰。在目标2中，我们将定义，在体内，有缺陷的IRES依赖的翻译在X-DC发病机制中的作用，利用遗传方法以及一种新的实时成像系统，以遵循IRES依赖的翻译。最后，在目标3中，我们将确定DKC 1 m核糖体未能利用体外重建系统启动蛋白质合成的分子步骤。抽象点。这些研究将为理解导致X-DC人类疾病的关键遗传和分子事件奠定更深的基础，并提供新的动物模型，这对于理解骨髓衰竭和癌症易感性的根本原因至关重要。