CELL TYPE-SPECIFIC CONTROL of GENE EXPRESSION by RIBOSOMAL PROTEIN ISOFORMS

核糖体蛋白亚型对基因表达的细胞类型特异性控制

• 批准号: 10056972
• 负责人: Adele Francis Xu
• 金额: $ 4.01万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10056972
• 关键词: Adult; Affect; Bioinformatics; Biological; Biological Models; Biological Process; Cell Proliferation; Cell physiology; Cells; Childhood; Computational Technique; Data; Defect; Development; Diamond-Blackfan anemia; Disease; Embryo; Epithelial; Epithelial Cells; Fractionation; Gene Expression; Gene Proteins; Genes; Genetic; Genetic Translation; Genome; Goals; Health; Hereditary hypotrichosis simplex; Human; Lactation; Mammalian Cell; Mammary gland; Mass Spectrum Analysis; Messenger RNA; Metabolism; Methods; Milk Proteins; Modeling; Molecular; Mus; Mutation; Open Reading Frames; Organelles; Pathology; Pattern; Physicians; Physiological; Population; Pregnancy; Proliferating; Protein Biosynthesis; Protein Isoforms; Protein Splicing; Proteins; Proteomics; Pseudogenes; RNA Splicing; Regulator Genes; Reporting; Ribosomal Proteins; Ribosomes; Role; Scientist; Structure; Techniques; Testing; Tissues; Transcript; Transgenic Mice; Translating; Translations; Variant; Work; autism spectrum disorder; cell type; combinatorial; computational pipelines; gene function; genome-wide; human disease; in vivo; insight; leukemia; mammary; neonate; novel; paralogous gene; protein expression; protein function; skeletal; transcriptome sequencing; transcriptomics

Project Abstract Mutations affecting protein components of the ribosome, an organelle essential in all nucleated cells for translating mRNA, underlie a growing list of congenital diseases. It remains unclear how various germline defects in the ubiquitous ribosome cause highly dissimilar and tissue-specific pathologies. Potential clues to this conundrum lie in recent reports that physiologic variation in ribosome composition can regulate translation of specific genes, often with tissue-specific effects. This novel gene regulatory paradigm suggests a myriad of mechanisms by which ribosomes affect development and cellular physiology that await discovery. Canonically, each ribosome contains 80 ribosomal proteins (RPs), most of which are each assumed to be encoded by a single gene. However, these RP genes have hundreds of little-understood splice variants, paralogs, and pseudogenes genome-wide, some of which have open reading frames that could produce proteins partly resembling canonical RPs. I hypothesize that certain splice variants, paralogs, and pseudogenes encode alternative RP isoforms that are expressed under specific biological conditions, and form distinct ribosomes with specialized roles in mRNA translation. Such a model could uncover combinatorially numerous possibilities for ribosome diversity, and reveal functions of many poorly understood ribosomal genes. To test my hypothesis, I will first characterize the RP paralog S27L as a paradigm model system of alternative RP function. My preliminary work suggests that, during lactation, mammary luminal epithelial cells undergo a dynamic switch by downregulating canonical RP S27 and upregulating its paralog S27L. This suggests that S27L-containing ribosomes may specialize in translating genes relevant to epithelial differentiation or high- volume protein synthesis. Second, I will develop the first systematic bioinformatic and proteomic pipeline to comprehensively investigate alternative RP expression across many cell types and developmental stages. The number of potential alternative RPs is vast, as is the number of biological conditions that may require their functions. There is therefore a need for a high-throughput approach using cell type- and developmental stage- resolved transcriptomic analysis to reveal conditions under which novel alternative RPs are expressed and incorporated into ribosomes. I will subsequently use ribosome fractionation and mass spectrometry to determine whether potential alternative RP transcripts are translated and incorporated into ribosomes in primary mouse tissues. Together, these orthogonal aims set a precedent for exploring the considerable potential impact of alternative RPs on development and health. Importantly, through this work I will gain diverse expertise in cutting-edge experimental methods, computational techniques, and scientific reasoning, advancing towards my goal as a physician-scientist to elucidate genetic mechanisms underlying human disease.

项目摘要 影响核糖体蛋白质成分的突变，核糖体是所有有核细胞中必不可少的细胞器 翻译 mRNA 是越来越多的先天性疾病的根源。目前尚不清楚各种种系如何 普遍存在的核糖体缺陷会导致高度不同的组织特异性病理。潜在线索 这个难题存在于最近的报道中，即核糖体组成的生理变化可以调节翻译 特定基因，通常具有组织特异性效应。这种新颖的基因调控范式提出了无数的 核糖体影响发育和细胞生理学的机制有待发现。按照规范， 每个核糖体含有 80 个核糖体蛋白 (RP)，其中大多数被认为是由 单基因。然而，这些 RP 基因有数百个鲜为人知的剪接变体、旁系同源物和 全基因组范围内的假基因，其中一些具有开放阅读框，可以部分产生蛋白质 类似于规范的 RP。我假设某些剪接变体、旁系同源物和假基因编码 在特定生物条件下表达并形成不同核糖体的替代 RP 亚型 在 mRNA 翻译中具有特殊作用。这样的模型可以揭示组合的多种可能性 核糖体多样性，并揭示许多人们知之甚少的核糖体基因的功能。为了测试我的 假设，我首先将 RP paralog S27L 描述为替代 RP 的范式模型系统 功能。我的初步工作表明，在哺乳期间，乳腺管腔上皮细胞经历了 通过下调规范 RP S27 并上调其旁系同源 S27L 进行动态切换。这表明 含有 S27L 的核糖体可能专门翻译与上皮分化或高分化相关的基因。 体积蛋白质合成。其次，我将开发第一个系统的生物信息学和蛋白质组学管道 全面研究多种细胞类型和发育阶段的替代 RP 表达。这 潜在的替代 RP 的数量是巨大的，可能需要其替代的生物条件的数量也是如此。 功能。因此，需要一种利用细胞类型和发育阶段的高通量方法 解析转录组分析以揭示新型替代 RP 的表达条件 并入核糖体。随后我将使用核糖体分离和质谱分析 确定潜在的替代 RP 转录本是否被翻译并整合到核糖体中 原代小鼠组织。这些相互正交的目标共同为探索相当大的领域开创了先例。 替代性移民计划对发展和健康的潜在影响。重要的是，通过这项工作我将获得多样化的 尖端实验方法、计算技术和科学推理方面的专业知识，推进 实现我作为一名医师科学家的目标，阐明人类疾病的遗传机制。