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: 10362546
• 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/10362546
• 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; 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 structure; Neurodegenerative Disorders; Neurons; Nuclear; Physical condensation; Polypyrimidine Tract-Binding Protein; Post-Transcriptional Regulation; 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; 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.

项目摘要/摘要 MIRA的应用是为了支持关于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的其他转录后步骤。我们将继续我们对两个家庭的研究 调节因子中包括多嘧啶结合蛋白和RBFox蛋白。我们将审查他们的 分子作用机制、生物学功能及其扩展调节程序的作用 在神经元发育和成熟的神经元功能。多种人类疾病，包括几种 神经退行性疾病，涉及RNA结合蛋白功能障碍和异常剪接调节。 为了开发这种疾病的治疗方法，我们需要更多地了解这种疾病的机制和 选择性剪接的生物学。我们将继续研究核型和细胞质的RBFox亚型。我们 将应用生物化学和基因组编辑的细胞系来研究八种RNA结合蛋白是如何 LasR复合体相互结合，以及组装的复合体如何与核RBFox相互作用进行调控 拼接。纯化的复合体的RNAseq分析和全基因组的iCLIP分析将绘制结合 与已知的RBFox结合位点相关的LasR亚基，以揭示LASR复合体内的RNA是如何 有条理的。我们将跟进最近对细胞质RBFox亚型的研究，以研究这些蛋白质是如何 调节编码重要突触蛋白的mRNAs的翻译和稳定性，如Vamp1。我们会 也继续我们对RBFox本质无序区域及其形成分子的能力的分析 凝析油。这些分析将扩展到LASR亚基中的IDR，以检查它们的同型和 异型相互作用及其凝聚在剪接调控中的作用。我们对人类社会的研究 PTBP1和PTBP2剪接抑制的机制和生物学将继续进行。我们将使用 早期工作中开发的生化方法和新的质谱学方法来检查 PTBP1抑制的外显子复合体的组装和结构及其阻断机制 高效的剪接体组装。我们将扩大我们对两个转变的生物影响的研究 神经元剪接调控：当PTBP1被PTBP取代时，在神经元发育早期诱导的一种调控， 一种发生在PTBP2在神经元成熟后期下调。特殊剪接的作用 PTBP计划内的开关将使用干细胞分化方案CRISPR进行检查 介导的基因编辑，以及整个转录组的表达和剪接分析。将RNAseq应用于 亚细胞组分，我们将表征由PTBP1控制的内含子保留事件，并研究 将RNA隔离在染色质上的机制。总之，这些研究将对 错综复杂的分子相互作用，调节剪接及其在人类疾病中的错误调节。