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

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 翻译信使核糖核酸是越来越多的先天性疾病的基础。目前还不清楚不同的生殖系是如何 无处不在的核糖体缺陷会导致高度不同的和组织特有的病理。潜在的线索 这一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 通常具有组织特异性效应的特定基因。这一新的基因调控范式表明了无数的 核糖体影响发育和细胞生理学的机制有待发现。经典地说， 每个核糖体包含80个核糖体蛋白(Rps)，其中大部分被认为是由一个 单基因。然而，这些rp基因有数百个鲜为人知的剪接变异体、平行对应体和 全基因组的假基因，其中一些有开放阅读框架，可以部分产生蛋白质 类似于规范的RPS。我假设某些剪接变体、并列基因和伪基因编码 在特定的生物条件下表达，并形成不同的核糖体的替代的RP异构体 在信使核糖核酸的翻译中扮演专门的角色。这样的模型可以揭示组合的无数可能性。 核糖体多样性，并揭示许多鲜为人知的核糖体基因的功能。测试我的 假设，我将首先将RP Paralog S27L描述为替代RP的范例模型系统 功能。我的初步工作表明，在哺乳期，乳腺管腔上皮细胞经历了一种 通过下调标准RP S27和上调其Paralog S27L来实现动态开关。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。 大量蛋白质合成。第二，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不同RP表达。这个 潜在的替代RP的数量是巨大的，可能需要它们的生物学条件的数量也是如此 功能。因此，需要一种使用细胞类型和发育阶段的高通量方法 解析转录分析以揭示表达新的替代RP的条件和 被整合到核糖体中。我随后将使用核糖体分离和质谱学来 确定潜在的替代RP转录本是否被翻译并整合到核糖体中 主要的小鼠组织。这些相互关联的目标共同开创了探索 替代方案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，不断进步 朝着我作为一名内科科学家的目标，阐明人类疾病背后的遗传机制。

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.