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

项目摘要 影响核糖体蛋白成分的突变，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基础。目前还不清楚各种生殖细胞 普遍存在的核糖体中的缺陷引起高度不同的和组织特异性的病理。潜在的线索 这个难题在于最近的报道，核糖体组成的生理变化可以调节翻译 特定的基因，通常具有组织特异性效应。这种新的基因调控模式表明， 核糖体影响发育和细胞生理的机制尚待发现。按照惯例， 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一个 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， 假基因是全基因组的，其中一些具有开放的阅读框架，可以部分产生蛋白质 类似于典型的RP。我假设某些剪接变异体，旁系同源物和假基因编码 在特定生物学条件下表达并形成不同核糖体的替代RP同种型 在mRNA翻译中扮演特殊角色。这样一个模型可以揭示组合的许多可能性 核糖体多样性，并揭示了许多知之甚少的核糖体基因的功能。测试我 假设，我将首先描述的RP paradise S27L作为一个范式模型系统的替代RP 功能我的初步工作表明，在哺乳期，乳腺腔上皮细胞经历了一个 通过下调经典RP S27和上调其paraminase S27L来动态切换。这表明 含S27 L的核糖体可能专门翻译与上皮分化或高分化相关的基因。 蛋白质合成量。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RP在许多细胞类型和发育阶段的替代表达。的 许多潜在的替代RP是巨大的，因为可能需要它们的生物条件的数量也是巨大的。 功能协调发展的因此，需要一种使用细胞类型和发育阶段的高通量方法， 解析转录组学分析，以揭示新的替代RP表达的条件， 整合到核糖体中。我随后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。总之，这些正交的目标为探索相当大的 替代品对发展和健康的潜在影响。重要的是，通过这项工作，我将获得不同的 尖端实验方法，计算技术和科学推理的专业知识， 我的目标是成为一名医学科学家，阐明人类疾病的遗传机制。

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.