Exploring the Role of Long Noncoding RNAs in Germinal Center B cells

生发中心(GC)反应是有效接种疫苗的基础，使亲和力成熟和 记忆力的发展。在迭代过程中，逐步选择亲和力更高的GC B细胞(GCBC)。在… 与此同时，GCBC的一小部分退出细胞周期并分化为长寿的后代--或者记忆 B细胞(MBC)或浆细胞(LLPC)。这些过程是通过集成来自 环境(例如，来自Ag、T细胞帮助)，它们又通过信号网络传递到细胞质和 细胞核，决定细胞行为和反应的地方。我们的实验室对这些过程进行了多次研究 水平，包括GC表面表型，信号的重新编程，基因表达和表观遗传 特定于GC的更改。许多实验室的工作揭示了复杂的转录因子(TF)网络 控制GC亲和力选择和分化的基因。而控制和诱导TF的信号显然是 重要的是，它们不太可能是完整的故事。RNA介导的控制是一层已经收到 少得多的关注。长的非编码RNA(LncRNAs)-它具有许多蛋白质的特征- 编码mRNA，如剪接、5‘帽和3’聚腺苷酸化--已经成为一类广泛而复杂的 调节分子负责调节细胞生物学的关键方面。除了监管 转录和翻译过程，lncRNAs参与表观遗传调控 染色体格局，以及许多其他不同的细胞过程，包括分子支架 和自动减支。人类基因组有比蛋白质编码RNA更多的lncRNA；然而，大多数 其中有哪些是未知的。有趣的是，许多lncRNA只在一个或几个组织中表达。最近，有几个 研究已经描述了在人类B细胞系中表达的lncRNAs，包括在GCBC中表达的。但是，相对的 由于其在基因组中的丰富表达，编码基因的lncrna仅在一个 水平较低，GCBC中尚无关于lncRNA的功能数据。这款R21是我们实验室的资金申请 研究这一新领域的初步工作是我们了解GC功能和基因调控的努力的一部分 网络。我们已经使用深度RNA-SEQ和严格的管道来鉴定推测的GC特异性LncRNAs， 通过定量聚合酶链式反应检测它们的表达，然后克隆和测序确定它们的结构。我们专注于 在这项建议中用遗传学和功能学的方法来理解三个最 有趣的lncRNAs(“GCLnc1、2和3”)。对于GCLnc1，我们已经生成了生殖系删除和 过表达系统：两者都有令人兴奋的初步表型。我们的目标是：1)全面描述B细胞 GCLnc1 KO小鼠的反应；2)产生GCLnc1、2和3的转录停止零等位基因，并 确定原代B细胞反应的初始表型；以及3)在GCBC中过表达每个GCLncRNA 我们实验室开发了一种新型的细胞转移系统，并测定了其体内的功能效应。我们将在以下时间完成此工作 与研究巨噬细胞中lncRNA功能的lncRNA专家曼尼杰·阿蒂亚南德博士合作。

The germinal center (GC) response is the basis for effective vaccination, enabling both affinity maturation and memory development. In an iterative process, higher affinity GC B cells (GCBC) are progressively selected. At the same time, a small fraction of GCBC exit cell cycle and differentiate into long-lived progeny—either memory B cells (MBC) or plasma cells (LLPC). These processes are controlled by integrating different signals from the environment (e.g. from Ag, T cell help) that in turn are transmitted via signaling networks to the cytoplasm and nucleus, where cellular behavior and responses are determined. Our lab has studied these processes at multiple levels, including GC surface phenotypes, reprogramming of signaling, and gene expression and epigenetic alterations that are GC-specific. The work of many labs has revealed complex transcription factor (TF) networks that control GC affinity selection and differentiation. While signals that control and induce TFs are clearly important, they are unlikely to be the complete story. RNA-mediated control is one layer that has received considerably less attention. Long non-coding RNAs (lncRNAs)—which share many characteristics of protein- coding mRNAs such as splicing, 5’ cap, and 3’ polyadenylation—have emerged as a broad, complex class of regulatory molecules responsible for modulating key aspects of cell biology. In addition to regulating the processes of transcription and translation, lncRNAs have been implicated in epigenetic regulation of the chromosomal landscape, as well as a host of other diverse cellular processes, including molecular scaffolding and sequestration. The human genome has more lncRNAs than protein-coding RNAs; yet, the function of most of these is unknown. Intriguingly, many lncRNAs are only expressed in one or a few tissues. Recently, a few studies have described lncRNAs expressed in the B cell lineage in human, including in GCBC. However, relative to their abundant representation in the genome, lncRNA encoding genes have been investigated at only a cursory level, and there are no functional data on lncRNA in GCBC. This R21 is a request for funding of our lab’s initial work to investigate this novel arena as part of our efforts to understand GC function and gene regulatory networks. We have used deep RNA-seq and a stringent pipeline to identify putative GC-specific lncRNAs, measured their expression via Q-PCR, then cloned and sequenced them to determine their structure. We focus in this proposal on using genetic and functional approaches to understand the function of three of the most interesting lncRNAs (“GCLnc1, 2 and 3”). For GCLnc1 we have already generated a germline deletion and an overexpression system: both have exciting preliminary phenotypes. Our Aims are: 1) To fully characterize B cell responses of the GCLnc1 KO mouse; 2) Generate transcriptional STOP null alleles of GCLnc1, 2 and 3 and determine initial phenotypes in primary B cell responses; and 3) Overexpress each GCLncRNA in GCBC using a novel cell transfer system developed in our lab and determine the functional effects in vivo. We will do this in collaboration with Dr. Maninjay Atianand, a lncRNA expert who works on lncRNA function in macrophages.