Role of Phosphorylation in RNA Binding Protein Function

项目摘要 许多RNA结合蛋白（RBP），调节选择性前mRNA剪接发生的基因家族， 具有高度一级和三级结构相似性的成员。但这些旁系同源体的组织并不重叠- 特异性表达模式和调节重叠和不同的靶外显子组，以引发组织特异性 拼接程序。这些旁系同源物如何实现组织特异性剪接模式尚不清楚，如果已知， 将有助于操纵基因表达以治疗组织特异性剪接相关疾病。在这 项目，我们建议研究翻译后磷酸化在组织特异性剪接中的作用， 多聚嘧啶道结合蛋白PTBP 1和PTPB 2的活性。PTBP 2的氨基酸序列为74%， 与PTBP 1相同。这两种蛋白质共享相似的结构域组织，识别并结合相同的蛋白质。 序列元件在相邻的靶外显子中，并且最常作为剪接阻遏物起作用。然而，PTBP 1 和PTBP 2具有不同的表达模式，在神经元发育和成熟中起关键作用。 神经元祖细胞表达PTBP 1，但在分化过程中，PTBP 1的水平下调， PTBP 2的表达上调。PTBP蛋白表达的这些变化改变了一组神经细胞的剪接， 外显子导致许多转录物的变化，这些转录物编码对轴突、树突发育至关重要的蛋白质 和突触的形成。因此，PTPB 1和PTPB 2表达的变化明显改变（从而调节） 神经元发育所需的剪接模式。然而，这些旁系同源物如何引出这些不同的 剪接结果是完全未知的。我们最近发现PTBP 1和PTBP 2是后 在剪接条件下被选择性磷酸化。PTBP 2具有更多的非重叠的不同位点 这些位点位于非结构化的N-末端和接头区域， 它们的序列同一性低于它们的RNA结合结构域。此外，PTBP 2明显磷酸化， PTBP 1中的氨基酸残基不保守，但在比人类低的物种中保持，这意味着它们是 在基因复制后获得/丢失，并且它们可能在PTBP 2剪接活性中起作用。这些发现 提示可逆磷酸化可能决定PTBP 1和PTBP 2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PTBP 2 RNA结合活性中的作用 目的2：确定连接区和磷酸化在PTBP 2剪接调控中的作用 目的3：确定参与PTBP 2神经元剪接调控的细胞信号通路 我们的研究将回答关于结构相关的旁系同源蛋白质 决定了不同的剪接结果，也揭示了神经元剪接程序是如何通过可逆的 磷酸化的RBP，如PTPB 2。 !

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. !