Role of E3 Ligase UBR5 in Alternative Splicing during B Cell Development and Activation

摘要 正常的B细胞发育是先天免疫和获得性免疫的重要组成部分。协调一项 许多分子机制都需要促进B细胞的发育、成熟、激活和 B细胞的存活。最近，泛素E3连接酶UBR5被发现在大量的 套细胞淋巴瘤患者2例，提示E3连接酶参与B细胞发育。UBR5是一个关键 DNA损伤修复、转录、翻译的调节因子，我们的数据表明，通过 剪接体。UBR5是一种Hect结构域连接酶，它含有半胱氨酸残基，负责 泛素转移到其底物上。为了阐明UBR5在B细胞成熟和激活中的作用 产生了一个条件突变，破坏了C-末端的Hect结构域。Hect结构域的丢失会导致 B细胞成熟受阻，B1和边缘B细胞亚群减少，以及 滤泡B细胞的表型改变和功能缺陷。蛋白质组学研究显示上调表达 B细胞中的剪接体蛋白缺乏UBR5的Hect结构域，并且UBR5与剪接相互作用 各种因素。为了进一步了解UBR5在调节B细胞激活和成熟中的功能作用以及 剪接体成分异常表达的后果，我们将定义UBR5在 生发中心的形成和激活(目标1)，决定泛素的独立功能与依赖功能 在B细胞中(AIM2)，并确定剪接体和改变的转录体在B细胞激活中的作用(目标3)。这些 研究将揭示UBR5通过调节关键转录本的选择性剪接在B细胞成熟中的新作用 在B细胞发育过程中。

ABSTRACT Proper B cell development is an essential part of innate and adaptive immunity. Coordination of a number of molecular mechanisms are required to facilitate B cell development, maturation, activation, and survival of B cells. Recently, the ubiquitin E3 ligase, UBR5 has been found mutated in a significant number of cases of patients with mantle cell lymphoma, implicating the E3 ligase in B cell development. UBR5 is a key regulator of DNA damage repair, transcription, translation, and our data suggests mRNA splicing via the spliceosome. UBR5 is a HECT domain ligase, which contains a cysteine residue that is responsible for ubiquitin transfer to its substrates. To elucidate the role of UBR5 in B cell maturation and activation we generated a conditional mutant disrupting the C-terminal HECT domain. Loss of the HECT domain leads to a block in maturation of B cells in the spleen with a reduction of B1 and marginal B cell subsets, as well as phenotypic alterations and functional defects of follicular B cells. Proteomic studies reveal up-regulation of spliceosome proteins in B cells lacking the HECT domain of UBR5, and that UBR5 interacts with splicing factors. To further understand the functional role of UBR5 in regulating B cells activation and maturation and the consequences of aberrant spliceosome component expression, we will define the role of UBR5 during germinal center formation and activation (Aim 1), determine the ubiquitin independent vs. dependent function in B cells (Aim2), and identify the role of spliceosome and altered transcripts in B cell activation (Aim 3). These studies will unravel a novel role of UBR5 in B cell maturation by regulating alternative splicing of key transcripts during B cell development.