Epigenetics in the extreme - investigating heritability driven by disordered RNA binding proteins in development and cancer

极端表观遗传学 - 研究发育和癌症中无序 RNA 结合蛋白驱动的遗传力

• 批准号: 10382634
• 负责人: Anupam Kumar Chakravarty
• 金额: $ 4.92万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2021-11-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10382634
• 关键词: Address; Adopted; Affinity; Alleles; Amino Acid Sequence; Amyloid; Amyloid fibers; Area; Behavior; Behavior Disorders; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Biophysics; Cancer Biology; Cancer cell line; Cell physiology; Cells; Cellular biology; Collaborations; Complex; Coupling; Data; Data Set; Development; Disease; Elements; Environment; Epigenetic Process; Exhibits; Flow Cytometry; Foundations; Gene Expression; Gene Expression Profiling; Generations; Genes; Genetic; Genome; Heritability; Homologous Gene; Human; Inherited; Investigation; Laboratories; Lead; Liquid substance; Malignant Neoplasms; Malignant neoplasm of ovary; Mammalian Cell; Measures; Medical center; Membrane; Memory; Mentors; Methodology; Methods; Microscopy; Molecular; Molecular Chaperones; Molecular Conformation; Nature; Nerve Degeneration; Oncogenic; Organelles; Organism; Pancreatic Adenocarcinoma; Pathology; Peptide Hydrolases; Phase; Phenotype; Physiological; Play; Prion Diseases; Prions; Property; Protein Conformation; Protein Overexpression; Proteins; Quantitative Genetics; RNA Binding; RNA-Binding Proteins; RNA-Protein Interaction; Reporter; Research; Research Personnel; Resistance; Resolution; Role; Specificity; Structure; Surface; System; TP53 gene; Techniques; Technology; Therapeutic Intervention; Training; Transcript; Translating; Variant; Yeasts; base; cancer type; career; cell type; exome; fascinate; fungus; insight; malignant breast neoplasm; mutant; nucleic acid binding protein; overexpression; prion-based; prion-like; programs; sea slug; solid state nuclear magnetic resonance; structural biology; therapeutic target; trait; transcriptome sequencing; transmission process; virtual; yeast prion

Heritable protein conformations, ‘prions’, were discovered as the cause of debilitating transmissible spongiform encephalopathies. Long considered to be a fascinating biological oddity, the recent discovery of prions in diverse organisms from fungi to sea slugs has led to the watershed realization that ‘protein-based genes’ are much more common than previously appreciated. We recently discovered that intrinsically disordered proteins can drive the formation of such protein-based molecular memories that can be epigenetically inherited over hundreds of generations without any alteration to its genome sequence. RNA binding proteins are particularly enriched in this dataset. An emergent area of study has demonstrated that disordered regions in RNA binding proteins are very common and often drives the formation of liquid-liquid de-mixing states critical for forming membrane-less organelles. My preliminary studies establish that disordered RNA binding proteins are uniquely poised to relay changes in environment to an altered cellular program. I will address the following aims during my remaining mentored training and initial independent research career: 1. To determine the biochemical and functional implication of intrinsically disordered regions of RNA binding proteins. I have developed a methodology to quantitatively investigate RNA-protein interactions across a genome. Coupling this platform with a robust system that I have developed to purify these intrinsically disordered RNA binding proteins, I intend to biochemically investigate the effect of intrinsically disordered regions on RNA binding proteins. Additionally, I will examine the effect of these disordered domains on phenotype using quantitative genetics and cell biology. Finally, I will extend the method to the human exome to interrogate human RBPs and their frequent oncogenic mutants. 2. To investigate the prion-like behavior of transient overexpression of disordered RNA binding protein ySmaug. Using high-throughput gene expression analysis, quantitative genetics and cell biology, I will examine the prion-like behavior of an evolutionarily ancient and highly disordered RNA binding protein ySmaug. 3. To establish the mechanism of assembly and transmission of prion-like RBPs. Using toolkits of biochemistry, microscopy and structural biology (NMR & EM methodologies), I will establish the mechanistic basis of assembly and transmission of these prion-like RBPs that exhibit canonical genetic hallmarks of prions but surprisingly do not form amyloids. 4. To investigate the impact of overexpression of disordered oncogenic proteins. Finally, I will harness a defining property of all known prions – protease resistance to examine the impact of three disordered nucleic acid binding proteins – p53 (one of its hotspot mutants is known to form amyloid fibers), SAMD4A (human homolog of ySmaug) and Musashi RNA binding proteins (Msi1 & Msi2) that is overexpressed in a variety of cancers and been attributed to be drivers of pancreatic adenocarcinomas. Following initial studies in yeast, I will investigate these proteins in their natural context – specifc mammalian cancer cell lines. The data obtained from this study will not only provide mechanistic insights into how higher- order heritable protein assemblies of RNA binding proteins are formed and heritably alter gene expression, but will also provide a framework for potential therapeutic intervention into a complex disease like cancer.

可遗传的蛋白质构象‘普里恩’被发现是导致衰弱的原因 传染性海绵状脑病。长期以来，它被认为是一种令人着迷的生物学怪异现象， 最近在从真菌到海参的各种生物中发现了普恩，这导致了 分水岭意识到以蛋白质为基础的基因比以前更常见 感激不尽。我们最近发现，本质上无序的蛋白质可以驱动这种形成 这种基于蛋白质的分子记忆可以从表观遗传上遗传超过数百 在不改变其基因组序列的情况下世代。RNA结合蛋白特别是 丰富了这一数据集。一个新兴的研究领域表明， RNA结合蛋白是一种非常常见的蛋白质，常导致液-液解混合的形成 对形成无膜细胞器至关重要的状态。我的初步研究表明 无序的RNA结合蛋白独特地准备将环境的变化传递给 改变了手机程序。在我剩下的指导培训期间，我将实现以下目标 初步独立研究生涯：1。生化和机能测定 暗示RNA结合蛋白的内在无序区域。我已经开发出一种 定量研究跨基因组的RNA-蛋白质相互作用的方法。偶联 这个平台有一个强大的系统，我开发这个系统是为了净化这些本质上的混乱 RNA结合蛋白，我打算用生物化学的方法研究其对内在紊乱的影响 RNA结合蛋白上的区域。此外，我将检查这些无序的影响 用数量遗传学和细胞生物学研究表型的区域。最后，我将扩展 方法从人外显子组询问人限制性商业惯例及其常见致癌突变体。 2.研究无序RNA瞬时过表达的类Pron行为 结合蛋白ySmaug。利用高通量的基因表达分析，数量遗传学 和细胞生物学，我将研究一种进化上古老而高度 无序RNA结合蛋白ySmaug。3.建立集结联动机制 Pron样限制性商业惯例的传播。使用生物化学、显微镜和结构学工具包 生物学(核磁共振和EM方法学)，我将建立组装和 这些具有普恩病毒典型遗传特征的类病毒限制性商业惯例的传播 令人惊讶的是，它不会形成淀粉样蛋白。4.调查过度表达的影响 无序的致癌蛋白。最后，我将利用所有已知的普里子的一个定义性质- 检测三种无序核酸结合蛋白--P53对蛋白酶抗性的影响 (它的一个热点突变体已知形成淀粉样纤维)，SAMD4A(人类同源物 YSmaug)和Musashi RNA结合蛋白(Msi1和MSI2)在各种 癌症，并被认为是胰腺癌的驱动因素。以下是初步研究 在酵母中，我将研究这些蛋白质在其自然上下文特定的哺乳动物癌细胞中的作用 台词。从这项研究中获得的数据不仅将提供机械上的洞察，了解- RNA结合蛋白的有序可遗传蛋白组合形成并遗传改变基因 表达，但也将提供一个框架，对复杂的潜在治疗干预 像癌症这样的疾病。