Role of RNA silencing in B cell development and function

描述（由申请人提供）：RNA沉默是一种新的主要生物学机制，由双链RNA触发，在信使RNA和染色质水平控制基因表达。最初在低等生物中发现，最近的结果表明它在哺乳动物发育和细胞分化中也起着至关重要的作用。基于RNA沉默可以影响B细胞发育的最新证据，以及其调节异常有助于B细胞淋巴瘤发生的强烈迹象，我们假设RNA沉默在B细胞发育的各个阶段起特定作用。后者是一个多阶段的过程，其特征在于染色体位点的复杂有序的相互作用，体细胞基因重排和突变依次发生，伴随着细胞增殖和静止的阶段，以及对拯救细胞免于凋亡的存活信号的持续需求。与某些成熟B细胞亚群的自我更新能力一起，其中一些特征与控制干细胞稳态和谱系定型的过程惊人地相似，已知这些过程是RNA沉默控制的主要目标。在所提出的实验中，我们将使用Cre/loxP介导的诱变将靶向突变引入B细胞发育的连续阶段。使用这种方法，我们将首先研究RNA沉默机制在多大程度上是正确的B细胞发育和功能所必需的，包括细胞执行适应性免疫应答和先天免疫功能（如抗原呈递给T细胞）的能力。在后一种情况下，树突状细胞（DC）将被包括在分析中。在这些实验中，我们将使用我们已经产生的条件d/cerallele，其已经证明Dicer是小鼠细胞中RNA沉默所需的。其次，我们将研究RNA沉默机制是否在B细胞发育过程中受到调节，如果是这样，我们将试图了解这种控制的基础。最后，我们将确定和功能特性的个别si/miRNAs的选择性和阶段特异性表达的B细胞和树突状细胞，使用生物信息学靶基因预测作为指导和小鼠的靶向诱变。总的来说，我们期待新的见解控制正常和恶性B细胞的发展和B细胞在适应性和先天性免疫反应的功能活性。鉴于B细胞在保护性免疫、自身免疫性疾病和淋巴瘤发生中的关键作用，我们预期这项工作将具有重大的医学意义。

DESCRIPTION (provided by applicant): RNA silencing is a novel, major biological mechanism controlling gene expression at the level of messenger RNA and chromatin, triggered by double-stranded RNA. Originally identified in lower organisms, recent results indicate that it also plays a crucial role in mammalian development and cell differentiation. Based on recent evidence that RNA silencing can affect B cell development, and strong indications that its dysregulation contributes to B cell lymphomagenesis, we hypothesize that RNA silencing plays specific roles at various stages of B cell development. The latter is a multi-stage process characterized by a sophisticated ordered interplay of chromosomal loci at which somatic gene rearrangements and mutation occur sequentially, accompanied by phases of cellular proliferation and quiescence and a continuous requirement for survival signals that rescue the cells from apoptosis. Together with the self-renewal capacity of certain mature B cell subsets, some of these features strikingly resemble processes that govern stem cell homeostasis and lineage commitment, known to be prime targets of control by RNA silencing. In the proposed experiments we will introduce targeted mutations into sequential stages of B cell development using Cre/loxP-mediated mutagenesis. Using this approach, we will first investigate the extent to which the RNA silencing machinery is required for proper B cell development and function, including the ability of the cells to execute adaptive immune responses and innate immune functions like antigen presentation to T cells. In the latter context, dendritic cells (DCs) will be included in the analysis. In these experiments we will use a conditional d/cerallele which we have generated, having demonstrated that Dicer is required for RNA silencing in mouse cells. Second, we will investigate whether the RNA silencing machinery is regulated during B cell development, and if so, try to understand the basis of such a control. Finally, we will identify and functionally characterize individual si/miRNAs that are selectively and stage-specifically expressed in B cells and DCs, using bioinformatic target gene predictions as a guide and targeted mutagenesis in mice. Overall, we expect new insights into the control of normal and malignant B cell development and the functional activity of B cells in adaptive and innate immune responses. Given the critical role of B cells in protective immunity, autoimmune diseases and lymphomagenesis, we anticipate this work will have significant medical relevance.