Role of rRNA Modifications in Dyskeratosis Congenita

描述(由申请人提供)： 这项建议的长期目标是阐明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，其特征是因骨髓衰竭而早期死亡，以及肿瘤易感性增加。在X-DC中发现的突变基因，先天性角化不良1(DKC1)，是一种假尿苷合成酶，通过将尿苷核苷酸(U)转化为假尿苷(Psi)，介导大小核糖体亚基的核糖体RNA(RRNA)转录后修饰。RRNA修饰在核糖体功能和蛋白质合成中的作用还知之甚少。我们已经培育出DKC1m亚型小鼠，它忠实地概括了X-DC的所有临床特征，是第一个严重再生障碍性贫血的小鼠模型，严重再生障碍性贫血是一种存在于以血细胞形成失败为特征的异质性疾病组中的危及生命的疾病。DKC1M小鼠也表现出癌症易感性的增加，其中超过50%的动物患上癌症和B细胞淋巴瘤。我们已经证明，DKC1M小鼠的原代细胞在rRNA假性尿路中存在损害，并在疾病发病前表现出核糖体活性降低。正如初步研究部分所概述的，我们已经成功地利用蛋白质组学策略来确定DKC1m细胞中翻译受损的mRNAs的子集。这些发现支持rRNA修饰在特定mRNAs的翻译起始中的作用，该mRNAs在其5‘UTR(5’非翻译区)中含有一个内部核糖体进入位点(IRES)。这项研究的目的是研究dyskerin和rRNA修饰在控制蛋白质合成中的作用。此外，利用分子和遗传学方法，我们将监测DKC1M小鼠体内的翻译调控，并从遗传学上测试关键靶标mRNAs的翻译损伤对X-DC病理特征的功能影响。在目标1中，我们将扩展我们的蛋白质组学策略来表征mRNAs，它依赖于dyskerin活性和rRNA修饰来启动翻译。在目标2中，我们将在体内利用遗传学方法和一种新的实时成像系统来跟踪IRES依赖的翻译，以确定缺陷的IRES依赖翻译在X-DC发病中的作用。最后，在目标3中，我们将确定DKC1m核糖体未能利用体外重组系统启动蛋白质合成的分子步骤(S)。保持抽象。总之，这些研究将为了解导致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，这将对了解骨髓衰竭和癌症易感性的根本原因至关重要。

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.