Antigens of the RNA-induced silencing complex in autoimmunity

描述（由申请人提供）：P.I.和合作者的实验室是第一个鉴定出称为GW体（GWB）的新型细胞质病灶的实验室，现在已知GW体富含在RNA干扰（RNAi）途径的效应臂中重要的人类自身抗原。通过表达克隆使用含有针对GW 182的抗体的人自身免疫血清来实现初始表征，GW 182是具有特异性定位于GWB的独特甘氨酸-色氨酸（GW）重复的182 KD蛋白自身抗原。GW 182被证明与选定的信使RNA（mRNA）和蛋白Ago 2相关，Ago 2是RNAi介导的mRNA加工中的关键酶。我们最近的数据表明，短干扰RNA（siRNA）和microRNA（miRNA）定位于GWB，有效的RNAi功能需要完整的GWB。综上所述，工作假设是GWB是通过siRNA/miRNA依赖性RNAi过程通过维持mRNA的稳定性和/或控制mRNA的降解来调节细胞质mRNA水平的新位点。迄今为止，已知针对GWB的人自身抗体具有识别GW 182、Ago 2和在P.I.的实验室。基于抗GW 182自身抗体与系统性红斑狼疮（SLE）和干燥综合征（SjS）这两种自身免疫性疾病的初步数据，设计了三个特定目的以帮助进一步表征GWB在正常和系统性风湿性疾病中的生物学意义。随着在SjS小唾液腺活检的某些病灶中观察到GW 182的过表达和在抗GW 182阳性自身免疫血清中抗52 kD SSA/Ro自身抗体的共表达，进一步发展了对SjS的兴趣。该研究的目的是探讨这些新的亚细胞病灶的生物学功能，这可能会对它们参与SLE和SjS的发病机制提供一些见解。具体目标1将解决GW 182、Ago 2和GWB中检测到的其他密切相关的自身抗原的生物学问题。具体目标2将在已建立的自身免疫性疾病小鼠模型中表征抗GWB抗体，并测试对GWB的自身免疫应答与靶器官中GWB表达的失调相关的假设，这可能导致RNAi缺陷。具体目标3将表征抗GWB抗体在人类疾病中的意义，并跟踪初步数据，即GW 182和SS-B/La（SjS和SLE中已知的主要自身抗原）在SjS唾液腺活检病灶中过表达。这些研究将有助于明确GWB在SLE和SjS发病机制中的生物学作用。动物模型将提供测试与这些靶向GWB的自身抗体的诱导相关的假设的手段。最近已经描述了miRNA在癌症中的意义，这项拟议的研究将解决miRNA失调是否可能在自身免疫性疾病中发挥作用。

DESCRIPTION (provided by applicant): The P.I. and collaborator's laboratories were the first to identify novel cytoplasmic foci known as GW bodies (GWBs), which are now known to be enriched with human autoantigens important in the effector arm of the RNA interference (RNAi) pathway. The initial characterization was achieved via expression cloning using a human autoimmune serum containing antibody to GW182, an 182KD protein autoantigen with unique glycine-tryptophan (GW) repeats that is specifically localized to GWBs. GW182 was shown to be associated with selected messenger RNAs (mRNAs) and the protein Ago2 which is the key enzyme in the RNAi mediated processing of mRNA. Our recent data showed that short interference RNAs (siRNAs) and microRNAs (miRNAs) are localized to GWBs and effective RNAi function requires intact GWBs. Taken together, the working hypothesis is that GWBs are novel sites regulating cytoplasmic mRNA levels via the siRNA/miRNA dependent RNAi process by maintaining stability and/or controlling degradation of mRNA. To date, human autoantibodies to GWBs are known to have overlapping subsets that recognize GW182, Ago2, and other autoantigens being characterized in the P.l.'s lab. Three Specific Aims have been designed to help further characterize the biological significance of GWBs in normal and in systemic rheumatic diseases including systemic lupus erythematosus (SLE) and Sjogren's syndrome (SjS) based on the preliminary data linking anti-GW182 autoantibody to these 2 autoimmune diseases. The interest in SjS is further developed with the observed overexpression of GW182 in certain foci of SjS minor salivary gland biopsies and the co- expression of anti-52kD SSA/Ro autoantibody in anti-GW182 positive autoimmune sera. The goal of the proposed study is to explore the biological functions of these novel subcellular foci which may shed some insight as to their involvement in the pathogenesis of SLE and SjS. Specific Aim 1 will address the biology of GW182, Ago2 and other closely associated autoantigens detected in GWBs. Specific Aim 2 will characterize anti-GWB antibodies in established mouse models of autoimmune diseases and test the hypothesis that autoimmune responses to GWBs are related to deregulation of GWB expression in target organs possibly resulting in defects in RNAi. Specific Aim 3 will characterize the significance of anti-GWB antibodies in human diseases and follow up on the preliminary data that GW182 and SS-B/La, a known major autoantigen in SjS and SLE, were overexpressed in foci of SjS salivary gland biopsies. The proposed studies will help define the biological role of GWBs in the pathogenesis of SLE and SjS. The animal models will provide the means to test hypotheses that are relevant to the induction of these autoantibodies targeting GWBs. Implication for mi RNA in cancer has been described recently and this proposed study will address whether there may be a role for miRNA deregulation in autoimmune diseases.