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

PTB 和 Rbfox 蛋白转录后基因调控机制

• 批准号: 10589873
• 负责人: Douglas L Black
• 金额: $ 77.81万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10589873
• 关键词: Alternative Splicing; Amyotrophic Lateral Sclerosis; Architecture; Binding; Binding Sites; Biochemical; Biochemistry; Biological; Biological Process; Biology; Cell Line; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Cytoplasm; Development; Disease; Epilepsy; Event; Exons; Family; Frontotemporal Dementia; Functional disorder; Gene Expression; Genes; Introns; Investigation; LasR protein; Maps; Mass Spectrum Analysis; Mediating; Methods; Molecular; Molecular Mechanisms of Action; Myotonic Dystrophy; Nervous System; Neurodegenerative Disorders; Neurons; Nuclear; Physical condensation; Polypyrimidine Tract-Binding Protein; Post-Transcriptional Regulation; Productivity; Protein Family; Protein Isoforms; Proteins; RNA; RNA Splicing; RNA-Binding Proteins; Reaction; Regulation; Repression; Role; Spinal Muscular Atrophy; Spliceosome Assembly Pathway; Subcellular Fractions; Synapses; Translations; Work; autism spectrum disorder; differentiation protocol; follow-up; genome editing; genome-wide; human disease; mRNA Precursor; mRNA Stability; nervous system disorder; neuron development; posttranscriptional; programs; stem cell differentiation; targeted treatment; transcriptome; transcriptome sequencing

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.

项目概要/摘要 该 MIRA 应用程序旨在支持 RNA 结合蛋白如何调节选择性剪接选择的研究 以及哺乳动物基因表达中的其他转录后步骤。我们将继续对两个家庭的研究 调节剂、聚嘧啶束结合蛋白和 Rbfox 蛋白。我们将检查他们的 分子作用机制、其生物学功能及其扩展调控程序的作用 神经元发育和成熟神经元功能。多种人类疾病，包括多种 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。 为了开发此类疾病的治疗方法，我们需要更好地了解其机制和作用 选择性剪接的生物学。我们将继续研究核和细胞质 Rbfox 亚型。我们 将应用生物化学和基因组编辑细胞系来检查八种RNA结合蛋白如何 LASR 复合物相互结合，以及组装后的复合物如何与核 Rbfox 相互作用来调节 拼接。纯化复合物的 RNAseq 分析和全基因组 iCLIP 分析将绘制 相对于已知 Rbfox 结合位点的 LASR 亚基，揭示 LASR 复合物内的 RNA 是如何变化的 有组织的。我们将跟进细胞质 Rbfox 亚型的最新研究，以检查这些蛋白质如何 调节编码重要突触蛋白（例如 Vamp1）的 mRNA 的翻译和稳定性。我们将 还继续我们对 Rbfox 本质无序区域及其形成分子的能力的分析 凝结物。这些分析将扩展到 LASR 亚基中的 IDR，以检查它们的同型性和 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 将继续研究 PTBP1 和 PTBP2 剪接抑制的机制和生物学。我们将使用 早期工作中开发的生化方法和新的质谱方法来检查 PTBP1 抑制的外显子复合物的组装和结构，并了解 PTBP1 如何阻断 高效的剪接体组装。我们将扩大对两个转变的生物学影响的调查 神经元剪接调节：当 PTBP1 被 PTBP2 取代时，在神经元发育早期诱导的一种调节， 一种是当 PTBP2 在神经元成熟后期下调时发生。特定拼接的作用 PTBP 程序中的开关将使用干细胞分化协议、CRISPR 进行检查 介导的基因编辑以及全转录组表达和剪接分析。将 RNAseq 应用于 亚细胞部分，我们将表征由 PTBP1 控制的内含子保留事件并检查 将 RNA 隔离在染色质上的机制。总而言之，这些研究将对 介导剪接调节及其在人类疾病中的错误调节的复杂分子相互作用。