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.

项目摘要/摘要 这种MIRA应用是为了支持RNA结合蛋白如何调节替代剪接中选择的研究 以及哺乳动物基因表达的其他转录后步骤。我们将继续研究两个家庭 调节剂，息肉酰胺氨基氨酸结合蛋白和rbfox蛋白。我们将检查他们 分子作用机制，其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中。多种人类疾病，包括几种 神经退行性疾病涉及RNA结合蛋白的功能障碍和异常的剪接调节。 要开发这种疾病的治疗方法，我们需要对机制和 替代剪接的生物学。我们将继续研究核和细胞质RBFOX同工型。我们 将采用生物化学和基因组编辑的细胞系来检查八种RNA结合蛋白如何 LASR复合物彼此结合，组装复合物如何与核RBFOX相互作用以调节 剪接。 RNASEQ对纯化的复合物和全基因组ICLIP分析的分析将映射的结合 LASR亚基相对于已知的RBFOX结合位点，以揭示LASR复合物中的RNA如何 有组织。我们将跟进细胞质RBFOX同工型的最新研究，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 还要继续我们对RBFOX固有无序区域的分析及其形成分子的能力 冷凝水。这些分析将扩展到LASR亚基中的IDR，以检查其同型和 异型相互作用及其在剪接调节中的凝结作用。我们对 PTBP1和PTBP2的剪接抑制机制和生物学将继续。我们将使用 在早期工作和新的质谱方法中开发的生化方法 外显子综合体的组装和体系结构被PTBP1抑制，并了解PTBP1如何阻止 生产剪接组装。我们将扩展对两种过渡的生物学影响的调查 神经元剪接调节：一种在PTBP2代替PTBP2时，一种在神经元发育的早期引起的， 当PTBP2在神经元成熟后期下调时，发生了。特定剪接的角色 PTBP程序中的开关将使用干细胞分化协议CRISPR检查 介导的基因编辑以及整个转录组表达和剪接分析。将RNASEQ应用于 亚细胞分数，我们将表征由PTBP1控制的内含子保留事件并检查 隔离染色质的RNA的机制。这些研究总共将产生对 复杂的分子相互作用，介导了剪接的调节及其在人类疾病中的不利影响。