ExRNA composition in SLE patients and factors influencing exRNP abundance and th

该项目的主要目的是确定系统性红斑狼疮（SLE）的可能触发因素， 评估在应激条件下积累的细胞外RNA（exRNA）和核糖核蛋白（RNP） 细胞质颗粒中的条件，可以刺激先天性免疫反应，并导致形成 自身抗体促进RNP颗粒形成或抑制其消退的因子的遗传突变 可能进一步影响它们的免疫刺激特性，包括大小、结构或局部浓度。 组织中核RBP和RNA加工因子的翻译后修饰，包括 磷酸化、泛素化、精氨酸二甲基化和蛋白酶切割，以及相关的 RNA及其特定的序列和结构伴随着不同的成熟阶段，可能是至关重要的 以区分正常与病理性和免疫原性RNP。 具体目标是： (1)用不同类型的抗体表征SLE患者的exRNA组成。研究是否 SLE患者永生化B细胞的exRNA组成反映了血清中检测到的变化 exRNA组成。分析细胞应激对永生化的免疫刺激特性的影响 在正常和应激条件下获得的B细胞裂解物或组织培养上清液，并鉴定 相关RNP的分馏实验。 (2)检查细胞外和细胞内RNA酶对exRNA丰度和组成的影响， 现有的小鼠模型，并调查是否有任何这些档案类似的exRNA组成的SLE 患者，认为RNA酶在SLE发病机制中的作用。 (3)开发人类细胞培养模型，用于压力相关的生化分离和表征 RNA颗粒。测试各种药物，以可重复地诱导特定的应激条件（例如氧化应激， 热休克或翻译停滞），并使用基于荧光测定来监测对RNA的动态影响 颗粒形成建立应用干扰素和细胞因子激活的测定法， 使用培养的细胞以及原代细胞在上述应激条件下进行。

The broad aim of this project is to identify possible triggers of systemic lupus erythematosus (SLE) and evaluate if extracellular RNA (exRNA) and ribonucleoproteins (RNPs), which accumulate under stress conditions in cytoplasmic granules, can stimulate innate immune responses and result in the formation of autoantibodies. Genetic mutations in factors facilitating RNP granule formation or inhibiting their resolution may further influence their immunostimulatory properties, including size, structure, or local concentration in tissues. Posttranslational modification of nuclear RBPs and RNA-processing factors including phosphorylation, ubiquitination, arginine dimethylation, and protease cleavage, as well as the associated RNAs and their specific sequences and structures accompanying various maturation stages, may be critical to distinguish normal from pathologic and immunogenic RNPs. The specific aims are: (1) Characterize the exRNA composition in SLE patients with distinct classes of antibodies. Examine if exRNA composition of cultured immortalized B cells from SLE patients mirrors variations detected in serum exRNA composition. Assay the effect of cellular stress on the immunostimulatory properties of immortalized B cell lysates or tissue culture supernatants obtained under normal and stress conditions, and identify relevant RNPs by fractionation experiments. (2) Examine the influence of extracellular and intracellular RNases on exRNA abundance and composition, in existing mouse models, and investigate if any of these profiles resembles the exRNA composition of SLE patients, arguing for a role of RNases in the pathogenesis of SLE. (3) Develop human cell culture models for the biochemical isolation and characterization of stress-related RNA granules. Test various drugs to reproducibly induce specific stress conditions (e.g. oxidative stress, heat shock or translational arrest) and use fluorescence-based assays to monitor dynamic effects on RNA granule formation. Establish assays for interferon and cytokine activation upon application of the aforementioned stress conditions using cultured cells as well as primary cells.