Roles of Ro60 RNPs in UV Stress and Cutaneous Autoimmunity

 描述(申请人提供)：Ro60自身抗原是一种环状RNA结合蛋白，已被发现在帮助细胞在紫外线(UV)照射后存活方面发挥进化保守的作用。基因敲除实验已经证明了Ro60在提高角质形成细胞紫外线应激后的存活率方面的重要性，但这是如何实现的仍不清楚。据报道，紫外线辐射会导致非编码RNA的结构变化，这可能会损害它们的功能，并增加重复序列衍生转录本的表达，这可能是细胞毒性的，或导致基因组不稳定。虽然其他几种RNA结合蛋白也被报道能提高紫外线应激后的存活率，但关于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。该项目的第一个目标是确定紫外线应激是否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的动态平衡。这些实验有可能发现细胞在紫外线照射后促进存活的一种新途径。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(RNPs)，如Ro60，在几种自身免疫性疾病中是自身抗体的靶标，有证据表明，这些RNPs的核酸部分对于通过激活Toll样受体(TLRs)启动炎症至关重要。紫外光被认为是SCLE炎症的主要触发因素，因此在UV应激期间与Ro60结合的RNA很可能是引发SCLE炎症的Ro60 RNPs的组成部分。因此，该项目的第二个目标是确定在紫外线应激下角质形成细胞中结合Ro60的RNA是否是有效的TLR激活剂。第一个目标是确定紫外线应激后人类角质形成细胞中与Ro60结合的一组RNA发生了多大程度的变化。这将通过体内交联、与抗Ro60抗体的免疫沉淀以及对与Ro60结合的RNA互补的cDNA的高通量测序(HITS-CLIP)来实现。HITS-CLIP将与传统的抗Ro60抗体和SCLE患者血清一起进行，以确定SCLE患者血清是否识别特定的Ro60 RNP子集。第二个目标是使用高通量测序来确定紫外线应激后Ro60靶基因的丰度，包括Ro60基因敲除和不基因敲除。这将表明Ro60结合是如何改变目标RNA的动态平衡的，并为缓解紫外线应激引起的RNA有害变化的途径提供了洞察。第三个目的是确定紫外线应激后与Ro60相关的RNA是否是血浆细胞样树突状细胞(PDCs)中TLRs的有效激活剂。在AIM 1中确定的RNA将以裸RNA或体外产生的含有Ro60的免疫复合体的形式呈现给PDCs，并检测它们激活TLR的能力。这一目标的实验应该确定最有可能导致SCLE炎症的一组RNA。鉴于它们可能在SCLE发病机制中的中心地位，这些RNA是潜在的治疗靶点。

 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.