Roles of Ro60 RNPs in UV Stress and Cutaneous Autoimmunity

Ro60 RNP 在紫外线应激和皮肤自身免疫中的作用

基本信息

批准号：
9060137
负责人：
MARCO E BOCCITTO
金额：
$ 5.8万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-05-01 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9060137
关键词：
Affect Antibodies Antigen-Antibody Complex Apoptosis Apoptotic Autoantibodies Autoantigens Autoimmune Diseases Autoimmune Process Autoimmunity Bacteria Binding Biological Assay Cell Survival Cell surface Cells Complementary DNA Coupled Cutaneous Cutaneous Lupus Erythematosus Data Dendritic Cells Dermatitis Disease Elements Endocytosis Exanthema Exoribonucleases Exposure to Genetic Transcription Genome Stability Genomic Instability Goals High-Throughput Nucleotide Sequencing Homeostasis Human Immunoprecipitation In Vitro Inflammation Inflammatory Interferons Knock-out Lupus Mammalian Cell Messenger RNA Modeling Monitor Mothers Mus Northern Blotting Nucleic Acids Pathogenesis Pathway interactions Patients Play Production Proteins RNA RNA Binding RNA I RNA-Binding Proteins Reporting Reverse Transcriptase Polymerase Chain Reaction Ribonucleoproteins Role Secondary to Serum Shapes Skin Stress Structure Sunlight Systemic Lupus Erythematosus Techniques Testing The Sun Toll-like receptors Toxic effect Transcript Transfer RNA UV Radiation Exposure UV induced Ultraviolet B Radiation Ultraviolet Rays Untranslated RNA base cell injury coping crosslink crosslinking and immunoprecipitation sequencing cytotoxic in vivo inhibitor/antagonist insight irradiation keratinocyte knock-down lupus cutaneous novel public health relevance research study skin disorder therapeutic target ultraviolet ultraviolet irradiation

项目摘要

DESCRIPTION (provided by applicant): The Ro60 autoantigen is a ring-shaped RNA binding protein that has been found to play an evolutionarily conserved role in in assisting cell survival following ultraviolet (UV) radiation. Knockout experiments have demonstrated the importance of Ro60 for enhancing survival after UV stress in keratinocytes, but how this is achieved is still unknown. UV radiation has been reported to cause structural changes in non-coding RNAs that may impair their function, and increase expression of repeat derived transcripts, which can be cytotoxic, or cause genomic instability. Although several other RNA binding proteins have also been reported to enhance survival after UV stress, little is known about how cells cope with changes in RNA integrity and composition induced by UV stress. The first goal of this project is to determine if UV stress alters the RNAs bound by Ro60 in keratinocytes, and determine how Ro60 binding influences the homeostasis of these RNAs. These experiments have the potential to uncover a novel pathway used by cells to promote survival after UV insult. Importantly, autoantibodies to Ro60 are strongly associated with the autoimmune dermatitis subacute cutaneous lupus erythematosus (SCLE). Ribonucleoproteins (RNPs), such as Ro60, are targeted by autoantibodies in several autoimmune disorders, and there is evidence that the nucleic acid portion of these RNPs is critical for initiating inflammation by activating toll-like receptors (TLRs). UV light is known to be a major trigger for inflammation in SCLE, therefore the RNAs bound to Ro60 during UV stress are likely to be components of Ro60 RNPs that initiate inflammation in SCLE. Thus, the second goal of this project is to determine if Ro60-bound RNAs in keratinocytes subjected to UV stress are potent TLR activators. The first aim is to determine the extent to which the set of RNAs bound to Ro60 is altered after UV stress in human keratinocytes. This will be achieved by in vivo crosslinking, immunoprecipitation with anti-Ro60 antibodies, and high-throughput sequencing of cDNAs complementary to the Ro60-bound RNAs (HITS-CLIP). HITS-CLIP will be performed with both traditional anti-Ro60 antibodies and SCLE patient sera, to determine whether SCLE patient sera recognizes a specific subset of Ro60 RNPs. The second aim is to use high throughput sequencing to determine the abundance of Ro60 targets after UV stress with and without Ro60 knockdown. This will indicate how Ro60 binding modifies the homeostasis of target RNAs, and provide insight into pathways that mitigate deleterious changes in RNA caused by UV stress. The third aim is to determine if RNAs associated with Ro60 after UV stress are potent activators of TLRs in plasmacytoid dendritic cells (PDCs). RNAs identified in Aim 1 will be presented to PDCs as either naked RNAs or in vitro generated Ro60-containing immune complexes and assayed for their ability to activate TLRs. The experiments in this aim should identify the set of RNAs that are most likely to contribute to inflammation in SCLE. Given their possible centrality in SCLE pathogenesis, these RNAs are potential therapeutic targets.

描述（由适用提供）：RO60自动抗原是一种环形RNA结合蛋白，已发现在紫外线（UV）辐射后在辅助细胞存活中起进化的作用。敲除实验证明了RO60在角质形成细胞中紫外应力后增强存活的重要性，但是如何实现这一点仍然未知。据报道，紫外线辐射会引起非编码RNA的结构变化，可能会损害其功能，并增加反复的衍生转录本的表达，这可能是细胞毒性的或引起基因组不稳定性。尽管还报道了其他几种RNA结合蛋白可以在紫外应激后增强存活率，但对于细胞如何应对RNA完整性和紫外应激诱导的组成的变化知之甚少。该项目的第一个目标是确定紫外线应力是否会改变角质形成细胞中RO60的RNA，并确定RO60结合如何影响这些RNA的稳态。这些实验有可能发现细胞在紫外线侮辱后促进生存的新途径。重要的是，RO60的自身抗体与自身免疫性皮炎亚急性皮肤红斑狼疮（SCLE）密切相关。核糖核蛋白（RNP），例如RO60，是几种自身免疫性疾病中的自身抗体的靶向，并且有证据表明，这些RNP的核酸部分对于通过激活Toll样受体（TLR）（TLRS）来启动注射至关重要。众所周知，紫外线是SCLE炎症的主要触发因素，因此在紫外应力期间与RO60结合的RNA可能是启动SCLE注入的RO60 RNP的组成部分。这是该项目的第二个目标是确定遭受紫外应力的角质形成细胞中的RO60结合RNA是否是潜在的TLR激活剂。第一个目的是确定人角质形成细胞紫外线应激后与RO60结合的RNA集合的程度。这将通过体内交联，与抗RO60抗体的免疫沉淀以及对RO60结合RNA（hits-CLIP）完整的CDNA的高通量测序。将使用传统的抗RO60抗体和SCLE患者血清进行hits-CLIP，以确定SCLE患者血清是否识别RO60 RNP的特定子集。第二个目的是使用高吞吐量测序来确定紫外线应力后有或没有RO60敲低的RO60目标的抽象。这将表明RO60结合如何改变靶RNA的体内稳态，并提供对途径减轻紫外应激引起的RNA有害变化的途径。第三个目的是确定紫外应激后与RO60相关的RNA是否是浆细胞类树突状细胞（PDC）中TLR的潜在活化剂。在AIM 1中鉴定出的RNA将以Naked RNA或体外产生的含RO60的免疫复合物的形式呈现给PDC，并分配了它们激活TLR的能力。此目标中的实验应确定最有可能导致SCLE炎症的RNA集。鉴于它们在SCLE发病机理中的可能中心性，这些RNA是潜在的治疗靶标。