Role of RNA silencing in B cell development and function

描述(申请人提供)：RNA沉默是一种新的、主要的生物学机制，由双链RNA触发，在信使RNA和染色质水平上控制基因的表达。最初发现于低等生物，最近的研究结果表明，它在哺乳动物的发育和细胞分化中也起着至关重要的作用。根据最近的证据，RNA沉默可以影响B细胞的发育，并且有强烈的迹象表明它的失调有助于B细胞的淋巴瘤化，我们假设RNA沉默在B细胞发育的不同阶段发挥着特定的作用。后者是一个多阶段的过程，以复杂有序的染色体位点相互作用为特征，体细胞基因重排和突变依次发生，伴随着细胞增殖期和静止期，以及对拯救细胞免于凋亡的生存信号的持续需求。结合某些成熟B细胞亚群的自我更新能力，这些特征中的一些惊人地类似于管理干细胞动态平衡和谱系承诺的过程，已知这两个过程是通过RNA沉默控制的主要目标。在拟议的实验中，我们将使用Cre/loxP介导的突变将靶向突变引入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B细胞正常发育和功能所必需的，包括细胞执行适应性免疫反应和先天免疫功能的能力，如将抗原递呈给T细胞。在后一种情况下，树突状细胞(DC)将包括在分析中。在这些实验中，我们将使用我们产生的有条件的d/cer等位基因，已经证明在小鼠细胞中RNA沉默是必需的。其次，我们将调查在B细胞发育过程中RNA沉默机制是否受到调控，如果是的话，试图了解这种调控的基础。最后，我们将以生物信息学的靶基因预测为指导，在小鼠中进行定向突变，鉴定在B细胞和DC中选择性和阶段特异性表达的单个si/miRNAs并对其进行功能表征。总体而言，我们期待在控制正常和恶性B细胞发育以及B细胞在适应性和先天免疫反应中的功能活动方面有新的见解。鉴于B细胞在保护性免疫、自身免疫性疾病和淋巴肿大中的关键作用，我们预计这项工作将具有重要的医学意义。

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.