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

摘要 适当的B细胞发育是先天免疫和适应性免疫的重要组成部分。配位 需要许多分子机制来促进B细胞发育、成熟、活化和增殖。 B细胞的存活。最近，已经发现泛素E3连接酶UBR 5在大量的人中发生突变。 例套细胞淋巴瘤患者，提示E3连接酶参与B细胞发育。UBR 5是一把钥匙 DNA损伤修复，转录，翻译的调节因子，我们的数据表明mRNA剪接通过 剪接体UBR 5是HECT结构域连接酶，其含有负责连接HECT的半胱氨酸残基。 泛素转移到其底物。为了阐明UBR 5在B细胞成熟和活化中的作用，我们 产生了破坏C-末端HECT结构域的条件突变体。HECT结构域的缺失导致 脾脏中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。蛋白质组学研究揭示了 缺乏UBR 5的HECT结构域的B细胞中的剪接体蛋白，并且UBR 5与剪接相互作用 因素为了进一步了解UBR 5在调节B细胞活化和成熟中的功能作用， 异常剪接体成分表达的后果，我们将定义UBR 5在 泛素中心的形成和激活（Aim 1），决定了泛素独立与依赖的功能 在B细胞中的作用（Aim 2），并鉴定剪接体和改变的转录物在B细胞活化中的作用（Aim 3）。这些 研究将揭示UBR 5通过调节关键转录物的选择性剪接在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.