Mechanisms of Post-transcriptional Gene Regulation by PTB and Rbfox Proteins

项目总结/摘要 这个MIRA应用程序是为了支持RNA结合蛋白如何调节选择性剪接的研究 以及哺乳动物基因表达中的其他转录后步骤。我们将继续研究两个家庭 调节剂，多嘧啶道结合蛋白和Rbfox蛋白。我们将检查他们的 分子作用机制，它们的生物学功能，以及它们的扩展调节程序的作用 在神经元发育和成熟神经元功能中。多种人类疾病，包括 神经退行性疾病涉及RNA结合蛋白的功能障碍和异常剪接调节。 为了开发治疗这些疾病的方法，我们需要更好地了解这些疾病的机制和治疗方法。 选择性剪接的生物学我们将继续我们的核和细胞质Rbfox亚型的研究。我们 将应用生物化学和基因组编辑的细胞系来研究细胞中的八种RNA结合蛋白是如何与基因组结合的。 LASR复合物相互结合，以及组装的复合物如何与核Rbfox相互作用以调节 拼接纯化的复合物的RNAseq分析和全基因组iCLIP分析将绘制结合蛋白的图谱。 LASR亚基相对于已知的Rbfox结合位点的结合，以揭示LASR复合物内的RNA是如何被结合的。 有组织的。我们将跟进最近的研究细胞质Rbfox亚型，以检查这些蛋白质如何 调节编码重要突触蛋白（如Vamp 1）的mRNA的翻译和稳定性。我们将 我们还继续分析了Rbfox的本征无序区及其形成分子的能力 冷凝物这些分析将扩展到LASR亚基中的IDR，以检查它们的同型性， 异型相互作用，以及它们的缩合在剪接调节中的作用。我们的研究 PTBP 1和PTBP 2的剪接抑制机制和生物学将继续进行。我们将使用 在早期工作中开发的生物化学方法和新的质谱方法来检查 PTBP 1抑制的外显子复合物的组装和结构，并了解PTBP 1如何阻断 生产性剪接体组装。我们将扩大我们的调查的生物影响的两个转变， 神经元剪接调节：当PTBP 1被PTBP 2取代时， 一种发生在PTBP 2在神经元成熟后期下调时。特定剪接的作用 PTBP项目中的开关将使用干细胞分化方案，CRISPR， 介导的基因编辑以及全转录组表达和剪接分析。应用RNAseq 亚细胞组分，我们将表征由PTBP 1控制的内含子保留事件，并检查 在染色质上隔离RNA的机制。总之，这些研究将产生新的认识， 在人类疾病中介导剪接调节及其失调的复杂分子相互作用。

PROJECT SUMMARY/ABSTRACT This MIRA application is to support studies of how RNA binding proteins regulate choices in alternative splicing and other posttranscriptional steps in mammalian gene expression. We will continue our studies of two families of regulators, the Polypyrimidine Tract Binding Proteins and the Rbfox proteins. We will examine their molecular mechanisms of action, their biological functions, and the roles of their extended regulatory programs in neuronal development and mature neuronal function. Multiple human diseases, including several neurodegenerative disorders, involve the dysfunction of RNA binding proteins and aberrant splicing regulation. To develop treatments for such disorders, we need greater understanding of both the mechanisms and the biology of alternative splicing. We will continue our studies of the nuclear and cytoplasmic Rbfox isoforms. We will apply biochemistry and genome edited cell lines to examine how the eight RNA binding proteins of the LASR complex bind with each other, and how the assembled complex interacts with nuclear Rbfox to regulate splicing. RNAseq analyses of purified complexes and genomewide iCLIP analyses will map the binding of LASR subunits relative to the known Rbfox binding sites to reveal how the RNA within the LASR complex is organized. We will follow up on recent studies of cytoplasmic Rbfox isoforms to examine how these proteins regulate the translation and stability of mRNAs encoding important synaptic proteins, such as Vamp1. We will also continue our analyses of the Rbfox intrinsically disordered region and its ability to form molecular condensates. These analyses will be extended to IDR’s in the LASR subunits to examine their homotypic and heterotypic interactions, and the role of their condensation in splicing regulation. Our studies of the mechanisms and biology of splicing repression by PTBP1 and PTBP2 will be continued. We will use biochemical methods developed in earlier work and new mass spectrometry approaches to examine the assembly and architecture of exon complexes repressed by PTBP1 and understand how PTBP1 blocks productive spliceosome assembly. We will extend our investigation of the biological impact of two transitions in neuronal splicing regulation: one induced early in neuronal development when PTBP1 is replaced with PTBP2, and one occurring when PTBP2 is downregulated late in neuronal maturation. The roles of particular splicing switches within the PTBP programs will be examined using stem cell differentiation protocols, CRISPR mediated gene editing, and whole transcriptome expression and splicing analyses. Applying RNAseq to subcellular fractions, we will characterize intron retention events controlled by PTBP1 and examine the mechanisms that sequester RNAs on chromatin. Altogether these studies will yield new understanding of the intricate molecular interactions that mediate the regulation of splicing and its misregulation in human disease.