Role of Phosphorylation in RNA Binding Protein Function

项目摘要 许多调节选择性前-mRNA剪接的RNA结合蛋白(RBP)以基因家族的形式出现 成员具有很高的一级和三级结构相似性。然而，这些Paralog有不重叠的组织- 特定的表达模式和调控重叠和不同的靶外显子集合以诱导组织特异性 拼接程序。这些对虾是如何实现组织特异性剪接模式的尚不清楚，如果已知， 将促进对基因表达的操纵，以治疗组织特异性剪接相关疾病。在这 项目中，我们建议研究翻译后磷酸化在组织特异性剪接中的作用 多嘧啶结合蛋白PTBP1和PTPB2的活性。PTBP2的氨基酸序列为74% 与PTBP1相同。这两种蛋白质共享相似的结构域组织，识别并结合到相同的结构域 相邻目标外显子中的序列元件和最常见的功能是剪接抑制物。然而，PTBP1 和PTBP2具有不同的表达模式，在神经元的发育和成熟过程中发挥着关键作用。 神经前体细胞表达PTBP1，但在分化过程中PTBP1水平下调，并且 PTBP2基因表达上调。PTBP蛋白表达的这些变化改变了一组神经元的剪接 外显子导致许多转录本的变化，这些转录本编码对轴突和树突的发育至关重要的蛋白质 以及突触的形成。因此，PTPB1和PTPB2表达的变化明显改变(并因此调节) 神经元发育所需的剪接模式。然而，这些比喻如何引出这些截然不同的 剪接的结果是完全未知的。我们最近发现PTBP1和PTBP2是后 在剪接条件下翻译磷酸化。PTBP2有更多非重叠的不同位点 比PTBP1更容易发生磷酸化，并且这些位点定位于非结构化N-末端和连接区， 它们比它们的RNA结合域具有更少的序列同一性。此外，PTBP2明显的磷酸化 残基在PTBP1中不保守，但在比人类更低的物种中保持，这意味着它们是 基因复制后获得/丢失，它们可能在PTBP2剪接活性中发挥作用。这些发现 提示可逆的磷酸化可能决定了PTBP1和PTBP2的组织特异性剪接活性。 我们的具体目标是检验这一假设。 目的1：确定磷酸化在PTBP2 RNA结合活性中的作用 目的2：确定连接区和磷酸化在PTBP2剪接调控中的作用 目的3：确定参与PTBP2神经元剪接调控的细胞信号通路 我们的研究将从根本上回答一些重要的问题，即结构上相关的平行蛋白质是如何 口述不同的剪接结果，并揭示神经元剪接程序是如何通过可逆的 限制性商业惯例的磷酸化，如PTPB2。 好了！

Project Summary Many RNA binding proteins (RBPs) that regulate alternative pre-mRNA splicing occur as gene families with members sharing high primary and tertiary structural similarity. Yet these paralogs have non-overlapping tissue- specific expression patterns and regulate over-lapping and distinct sets of target exons to elicit tissue-specific splicing programs. How these paralogs achieve tissue-specific splicing patterns is not understood, and if known, would facilitate the manipulation of gene expression to treat tissue-specific splicing related diseases. In this project, we propose to investigate the role of post-translational phosphorylation in the tissue-specific splicing activities of polypyrimidine tract binding proteins PTBP1 and PTPB2. The amino acid sequence of PTBP2 is 74% identical to that of PTBP1. The two proteins share a similar domain organization, recognize and bind to the same sequence elements in adjacent target exons and most often function as splicing repressors. However, PTBP1 and PTBP2 have distinct expression patterns that play a critical role in neuronal development and maturation. Neuronal progenitor cells express PTBP1, but during differentiation the level of PTBP1 is down-regulated, and that of PTBP2 is up-regulated. These changes in PTBP protein expression alter the splicing of a set of neuronal exons leading to changes in many transcripts that code for proteins critical for development of axons, dendrites and the formation of synapses. Thus, changes in PTPB1 and PTPB2 expression clearly alter (and thus regulate) the patterns of splicing required for neuronal development. However, how these paralogs elicit these distinct splicing outcomes is completely unknown. We recently discovered that PTBP1 and PTBP2 are post- translationally phosphorylated under splicing conditions. PTBP2 has many more non-overlapping distinct sites of phosphorylation than PTBP1 and these sites are localized to the unstructured N-terminal and linker regions, which share less sequence identity than their RNA binding domains. Moreover, PTBP2 distinct phosphorylated residues are not conserved in PTBP1, yet are maintained in lower species than humans implying they were acquired/lost after gene duplication and that they may play a role in PTBP2 splicing activity. These findings suggest that reversible phosphorylation might dictate the tissue-specific splicing activities of PTBP1 and PTBP2. Our specific aims are to test this hypothesis. Aim 1: Determine the role of phosphorylation in PTBP2 RNA binding activity Aim 2: Determine the role of linker regions and phosphorylation in PTBP2 splicing regulation Aim 3: Determine cell signaling pathways involved in PTBP2 neuronal splicing regulation Our studies would answer fundamentally important questions about how structurally related paralogous proteins dictate different splicing outcomes and also reveal how the neuronal splicing program is modulated via reversible phosphorylation of RBPs such as PTPB2. !