RNA quality control and environmental stress

描述（由申请人提供）：尽管不能降解细胞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，截短和错误折叠的rRNA，tRNA和其他非编码RNA可以由基因突变，转录错误和加工错误产生。RNA也会被阳光照射和其他环境损伤所破坏。此外，由于30-50%的哺乳动物基因组由重复元件组成，包括活性逆转录转座子及其许多保持转录活性的截短和分歧亲属，因此必须识别和降解来自这些元件的转录物。大多数RNA衰变的研究都是在酵母中进行的，其中称为外泌体的核酸外切酶复合物是非编码RNA衰变的主要贡献者。其他途径可能有助于后生动物，因为当外泌体亚基在酵母中突变时发生的许多异常非编码RNA的积累在亚基从动物细胞中耗尽时尚未观察到。该项目的目的是描述一种新的途径，通过该途径在哺乳动物细胞和一些细菌中处理异常的非编码RNA。重点是Ro 60 kD自身抗原，SLE和干燥综合征患者免疫应答的重要靶点，可能参与几种临床后遗症。缺乏Ro的小鼠会发展出类似于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很重要。Ro是环状的，并且结合新合成的错误折叠的RNA，使得它们的单链3'端从Ro中心腔突出。在细胞质中，Ro结合称为Y RNA的非编码RNA，其调节Ro的亚细胞分布。在至少一种细菌中，Ro和Y RNA与环形外切核酸酶多核苷酸磷酸化酶（PNIPs）形成复合物，其在营养胁迫期间的rRNA降解中起作用。我们的第一个目标是测试的假设，基于电子显微镜的细菌复合物，单链RNA线程通过Ro环进入PNTR腔和Y RNA支架复合物。我们的第二个目的是确定Ro在多大程度上调节小鼠细胞中异常转录本的表达。我们将测试的假设，在小鼠胚胎干细胞中，Ro的功能在转录的重复元件的衰变的基础上，Ro的结合RNA的交联。我们的第三个目标是确定Ro暴露于紫外线照射的哺乳动物细胞和细菌后的直接目标。总之，这些研究应该阐明一种新的机制，调节RNA衰变的核酸外切酶，阐明了临床上重要的RNA结合蛋白在哺乳动物RNA代谢中的作用，并可能产生洞察非编码RNA代谢途径的作用，在适应RNA群体在压力下，一个知之甚少，但可能是维持细胞内稳态的重要组成部分。 公共卫生相关性：针对称为Ro 60 kDa自身抗原的RNA结合蛋白的抗体与患有系统性红斑狼疮的成人的日光敏感性皮肤病变以及具有这些抗体的母亲的婴儿的类似皮疹和永久性心脏缺陷相关。在从垂死或应激细胞释放后，RO结合的RNA被提出通过与抗体和激活控制引起组织损伤的分子的产生的受体相关联来促成这些症状。研究Ro在细胞中的功能并识别Ro结合的RNA可能使设计特异性靶向Ro或其相关RNA分子的药物成为可能。

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. PUBLIC HEALTH RELEVANCE: Antibodies against an RNA-binding protein called the Ro 60 kDa autoantigen are associated with sunlight- sensitive skin lesions in adults with systemic lupus erythematosus and with similar skin rashes and permanent heart defects in babies of mothers with these antibodies. After release from dying or stressed cells, Ro-bound RNAs are proposed to contribute to these symptoms by associating with the antibodies and activating receptors that control the production of molecules that cause tissue damage. Studying how Ro functions in cells and identifying Ro-bound RNAs may make it possible to design drugs that specifically target either Ro or its associated RNA molecules.