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

• 批准号: 8835566
• 负责人: MARCO E BOCCITTO
• 金额: $ 5.42万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 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/8835566
• 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; 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; Lupus Erythematosus, Cutaneous, Subacute; Mammalian Cell; Messenger RNA; Modeling; Monitor; Mothers; Mus; Northern Blotting; Nucleic Acids; Pathogenesis; Pathway interactions; Patients; Play; Production; Proteins; RNA; RNA Binding; 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; lupus cutaneous; novel; public health relevance; research study; skin disorder; therapeutic target; ultraviolet; ultraviolet irradiation

 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（由申请人提供）：Ro60自身抗原是一种环形RNA结合蛋白，已发现其在协助紫外线（UV）辐射后细胞存活方面发挥进化保守的作用。敲除实验已经证明了 Ro60 对于增强角质形成细胞在紫外线应激后的存活率的重要性，但如何实现这一点仍不清楚。据报道，紫外线辐射会引起非编码 RNA 的结构变化，从而可能损害其功能，并增加重复衍生转录本的表达，这可能具有细胞毒性，或导致基因组不稳定。尽管也有报道称其他几种 RNA 结合蛋白可以提高紫外线应激后的存活率，但人们对细胞如何应对紫外线应激引起的 RNA 完整性和组成的变化知之甚少。该项目的首要目标是确定紫外线应激是否会改变角质形成细胞中 Ro60 结合的 RNA，并确定 Ro60 结合如何影响这些 RNA 的稳态。这些实验有可能揭示细胞在紫外线损伤后促进存活的新途径。重要的是，Ro60 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，是多种自身免疫性疾病中自身抗体的靶标，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) 引发炎症至关重要。众所周知，紫外线是 SCLE 炎症的主要触发因素，因此在紫外线应激期间与 Ro60 结合的 RNA 很可能是引发 SCLE 炎症的 Ro60 RNP 的组成部分。因此，该项目的第二个目标是确定遭受紫外线应激的角质形成细胞中 Ro60 结合的 RNA 是否是有效的 TLR 激活剂。第一个目标是确定与 Ro60 结合的 RNA 组在人类角质形成细胞中受到紫外线应激后发生的改变程度。这将通过体内交联、抗 Ro60 抗体免疫沉淀以及与 Ro60 结合 RNA 互补的 cDNA 的高通量测序 (HITS-CLIP) 来实现。将使用传统抗 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。第二个目标是使用高通量测序来确定在有和没有 Ro60 敲低的情况下 UV 胁迫后 Ro60 靶标的丰度。这将表明 Ro60 结合如何改变靶标 RNA 的稳态，并深入了解减轻紫外线应激引起的 RNA 有害变化的途径。第三个目标是确定紫外线应激后与 Ro60 相关的 RNA 是否是浆细胞样树突状细胞 (PDC) 中 TLR 的有效激活剂。目标 1 中鉴定的 RNA 将作为裸露 RNA 或体外生成的含 Ro60 的免疫复合物呈递给 PDC，并分析其激活 TLR 的能力。此目的的实验应确定最有可能导致 SCLE 炎症的一组 RNA。鉴于它们在 SCLE 发病机制中可能的中心地位，这些 RNA 是潜在的治疗靶点。