RNA quality control and environmental stress

描述（由申请人提供）：虽然不能降解细胞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大多数异常rna的分子和途径知之甚少。大多数RNA不编码蛋白质，基因突变、转录错误和加工错误会产生截短和错误折叠的RNAs、tRNAs和其他非编码RNA。rna也会受到阳光照射和其他环境损害的破坏。此外，由于30-50%的哺乳动物基因组由重复元件组成，包括活跃的反转录转座子及其许多截断和分化的亲属，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，其中被称为外泌体的外切酶复合物是非编码RNA衰变的主要贡献者。其他途径可能在后生动物中起作用，因为当酵母中的外泌体亚基发生突变时，未观察到当亚基从动物细胞中耗尽时，会发生大量异常非编码rna的积累。这个项目的目的是表征异常非编码rna在哺乳动物细胞和一些细菌中处理的新途径。研究的重点是Ro 60kd自身抗原，这是SLE和干燥综合征患者免疫应答的重要靶点，也是多种临床后遗症的可能参与者。缺乏Ro的小鼠会发生类似SLE患者的自身免疫性疾病，而Ro对于哺乳动物细胞和至少一种真细菌在紫外线照射后的存活至关重要。Ro呈环状，结合新合成的错误折叠rna，使它们的单链3'端从Ro中心腔中伸出。在细胞质中，Ro与称为Y rna的非编码rna结合，Y rna调节Ro的亚细胞分布。在至少一种细菌中，Ro和Y RNA与环状核酸外切酶，多核苷酸磷酸化酶（PNPase）形成复合物，在营养胁迫下作用于rRNA降解。我们的第一个目标是测试假设，基于细菌复合体的电子显微镜，单链RNA穿过Ro环进入PNPase腔，Y RNA支撑复合体。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物的表达。我们将基于Ro与小鼠胚胎干细胞中结合的rna的交联来验证这一假设，即Ro在重复元件转录本的衰变中起作用。我们的第三个目标是确定在哺乳动物细胞和细菌暴露于紫外线照射后Ro的直接目标。总之，这些研究应该阐明通过外切酶调节RNA衰变的新机制，阐明临床重要的RNA结合蛋白在哺乳动物RNA代谢中的作用，并可以深入了解非编码RNA代谢途径在逆境中适应RNA群体中的作用，这是一个知之甚少但可能是维持细胞稳态的重要部分。

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.