RNA quality control and environmental stress

描述(申请人提供)：尽管未能降解细胞RNA会导致神经系统疾病和自身免疫性疾病，如系统性红斑狼疮(SLE)，但对哺乳动物细胞中降解最异常RNA的分子和途径知之甚少。大多数RNA不编码蛋白质，截短和错误折叠的rRNAs、tRNAs和其他非编码RNAs可以由基因突变、转录错误和加工错误产生。日光照射和其他环境侮辱也会破坏RNA。此外，由于30%-50%的哺乳动物基因组由重复元件组成，包括活性反转录转座子及其许多截断的和保持转录活性的不同近亲，这些元件的转录本必须被识别和降解。大多数关于RNA衰变的研究都是在酵母中进行的，在酵母中，一种被称为外切体的外切酶复合体是非编码RNA衰变的主要贡献者。其他途径可能在后生动物中起作用，因为当酵母中的外切体亚单位发生突变时，当这些亚单位从动物细胞中耗尽时，并没有观察到大量异常的非编码RNA的积累。这个项目的目的是描述一种在哺乳动物细胞和一些细菌中处理异常非编码RNA的新途径。焦点是Ro 60kD自身抗原，它是SLE和干燥综合征患者免疫反应的重要靶点，可能参与了几种临床后遗症。缺乏Ro的小鼠会患上一种类似于SLE患者的自身免疫性疾病，在哺乳动物细胞和至少一种真细菌中进行紫外线照射后，Ro对生存很重要。Ro是环状的，与新合成的错误折叠的RNA结合，使其单链3‘端从Ro中心空腔伸出。在细胞质中，Ro结合非编码RNA，称为Y RNAs，调节Ro的亚细胞分布。在至少一种细菌中，Ro和Y RNA形成一个具有环状核酸外切酶的复合体，即多核苷酸磷酸化酶(PNPase)，该酶在营养胁迫期间对rRNA降解起作用。我们的第一个目标是基于细菌复合体的电子显微镜来验证这一假设，即单链RNA通过Ro环进入PNPase空腔，而Y RNA是复合体的支架。我们的第二个目标是确定Ro在多大程度上调节小鼠细胞中异常转录本的表达。我们将基于Ro与小鼠胚胎干细胞中结合的RNA的交叉连接来检验这一假设，即Ro在重复元素转录物的衰退中发挥作用。我们的第三个目标是确定Ro在哺乳动物细胞和细菌暴露于紫外线照射后的直接靶点。总之，这些研究将阐明一种通过核酸外切酶调节RNA衰退的新机制，阐明一种临床上重要的RNA结合蛋白在哺乳动物RNA代谢中的作用，并可能深入了解非编码RNA代谢途径在适应应激过程中RNA群体的作用，这是一个知之甚少但可能是维持细胞内稳态的重要部分。

DESCRIPTION (provided by applicant): Although the failure to degrade cellular RNAs contributes to neurologic disorders and to autoimmune diseases such as systemic lupus erythematosus (SLE), little is known of the molecules and pathways that degrade most aberrant RNAs in mammalian cells. Most RNA does not code for proteins, and truncated and misfolded rRNAs, tRNAs and other noncoding RNAs can be generated by gene mutations, transcriptional errors, and processing mistakes. RNAs can also be damaged by sunlight exposure and other environmental insults. Also, since 30-50% of mammalian genomes consists of repetitive elements, including active retrotransposons and their many truncated and divergent relatives that remain transcriptionally active, transcripts from these elements must be recognized and degraded. Most studies of RNA decay have been carried out in yeast, where an exonuclease complex known as the exosome is a major contributor to noncoding RNA decay. Additional pathways likely contribute in metazoans, as the accumulation of numerous aberrant noncoding RNAs that occurs when exosome subunits are mutated in yeast has not been observed when the subunits are depleted from animal cells. The objective of this project is to characterize a novel pathway by which aberrant noncoding RNAs are handled in mammalian cells and some bacteria. The focus is on the Ro 60 kD autoantigen, an important target of the immune response in patients suffering from SLE and Sjogren's syndrome and a likely participant in several clinical sequelae. Mice lacking Ro develop an autoimmune disease that resembles SLE in patients, and Ro is important for survival after UV irradiation in mammalian cells and at least one eubacterium. Ro is ring-shaped and binds newly synthesized misfolded RNAs such that their single-stranded 3' ends protrude from the Ro central cavity. In the cytoplasm, Ro binds noncoding RNAs called Y RNAs that regulate the subcellular distribution of Ro. In at least one bacterium, Ro and a Y RNA form a complex with a ring-shaped exonuclease, polynucleotide phosphorylase (PNPase), that functions in rRNA degradation during nutritional stress. Our first aim is to test the hypothesis, based on electron microscopy of the bacterial complex, that single-stranded RNA threads through the Ro ring into the PNPase cavity and that the Y RNA scaffolds the complex. Our second aim is to determine the extent to which Ro modulates expression of aberrant transcripts in mouse cells. We will test the hypothesis, based on cross-linking of Ro to bound RNAs in mouse embryonic stem cells, that Ro functions in the decay of transcripts from repetitive elements. Our third aim is to identify the direct targets of Ro following exposure of mammalian cells and bacteria to UV irradiation. Together, these studies should elucidate a novel mechanism for modulating RNA decay by exonucleases, illuminate the role of a clinically important RNA-binding protein in mammalian RNA metabolism, and could yield insights into the roles of noncoding RNA metabolic pathways in adapting RNA populations during stress, a poorly understood but likely important part of maintaining cellular homeostasis.