Control of ribosomal function in cells of the hematopoietic system

项目概要/摘要 翻译是一个动态的过程，由包括mRNA在内的多种因素赋予高度特异性 转录物序列、翻译机制的组成和细胞环境。一个惊人的示范， 这种特异性是组织特异性的（例如颅面异常、骨髓衰竭），而不是全局性的 在人类核糖体病中观察到的发育缺陷中， 因素此外，核糖体病驱动的骨髓衰竭的特征是选择性的，而不是全球性的， mRNA翻译的缺陷。因此，甚至核糖体的组成也会影响mRNA的翻译。 以组织特异性方式提高效率。这些观察结果表明， 在造血系统中的翻译;阐明这种调节的机制，因此是必不可少的， 了解疾病状态下造血细胞的发育、功能和功能障碍。 因此，我的长期研究目标是阐明翻译控制的机制， 在正常免疫功能期间和疾病状态下的血淋巴样细胞，重点是核糖体相关的 蛋白质（RAP）-一类研究较少的蛋白质，与核心核糖体蛋白质发生物理相互作用， 调节特定mRNA的翻译效率。我的主要假设是，在免疫细胞中， 对感染的快速反应，炎症刺激可能诱导RAP与核糖体的动态相互作用， 它们可以影响特定mRNA翻译效率的快速变化， 免疫调节的后果。在以前的工作中，我已经定义了核糖体结合RAP的全部功能， 体外骨髓来源的巨噬细胞。在本提案的目标1中，我将使用全球性的、无偏见的蛋白质组学， TLR刺激后活化巨噬细胞中RAP-核糖体相互作用的时间分布。在目标2中，我 将使用CRISPR基因编辑，核糖体分析和细胞因子分泌测定来1）鉴定mRNA靶点， 受我已经确定的两种RAP（Rnf 213和Helz 2）以及其他RAP的特异性调节 目的1中鉴定的，和2）确定这些RAP在巨噬细胞炎症反应中的功能。 作为临床病理学家和血液病理学家，完成了医学博士培训和临床培训， 我正在申请K38奖，以支持我成为一名独立医生调查员的目标。加州大学旧金山分校 特殊的培训环境，特别是在免疫学和蛋白质稳定领域，使其成为理想的场所 让我继续努力我的职业发展计划的关键要素包括利用专业知识 我的主要导师，核糖体生物学领域的先驱Davide Ruggero博士和我的共同导师， 免疫专家Jason Cyster和血液病理诊断专家Karthik Ganapathi博士; 医生-科学家顾问的指导;数据科学和研究伦理的课程;以及其他专业 发展活动。总之，我衷心相信这个职业发展计划将提供一个强大的 最终建立一个独立的研究计划。

PROJECT SUMMARY/ABSTRACT Translation is a dynamic process with a high degree of specificity conferred by multiple factors including mRNA transcript sequence, composition of the translation machinery, and cellular milieu. A striking demonstration of this specificity is the tissue-specific (e.g. craniofacial anomalies, bone marrow failure) rather than global nature of the developmental defects seen in human ribosomopathies caused by congenital mutations in ribosomal factors. Moreover, ribosomopathy-driven bone marrow failure is characterized by selective, rather than global, defects in mRNA translation. Thus, even the composition of the ribosome can affect mRNA translational efficiency in a tissue-specific manner. These observations point to a particularly nuanced regulation of translation in the hematopoietic system; elucidating the mechanisms of this regulation is therefore essential for understanding hematopoietic cell development, function, and dysfunction in disease states. As such, my long-term research goal is to elucidate mechanisms of translational control in hematolymphoid cells during normal immune function and in disease states, focusing on ribosome-associated proteins (RAPs)—a poorly studied class of proteins which physically interact with the core ribosomal proteins to tune the translational efficiency of specific mRNAs. My central hypothesis is that, in immune cells poised for rapid responses to infection, inflammatory stimuli may induce dynamic interactions of RAPs with ribosomes, where they may effect rapid changes in the translational efficiency of specific mRNAs with important immunoregulatory consequences. In previous work, I have defined the repertoire of ribosome-bound RAPs in in vitro bone marrow-derived macrophages. In Aim 1 of this proposal, I will use global, unbiased proteomics to temporally profile RAP-ribosome interactions in activated macrophages following TLR stimulation. In Aim 2, I will use CRISPR gene editing, ribosome profiling, and cytokine secretion assays to 1) identify mRNA targets that are specifically regulated by two RAPs I have already identified, Rnf213 and Helz2, as well as other RAPs identified in Aim 1, and 2) determine the function of these RAPs in the macrophage inflammatory response. Having completed MD-PhD training and clinical training as a clinical pathologist and hematopathologist, I am applying for the K38 Award to support my goal of becoming an independent physician investigator. UCSF’s exceptional training environment, especially in the areas of immunology and proteostasis, make it an ideal place for me to pursue my efforts. Critical elements of my career development plan include leveraging the expertise of my primary mentor Dr. Davide Ruggero, a pioneer in the field of ribosome biology, and my co-mentors Dr. Jason Cyster, an expert immunologist, and Dr. Karthik Ganapathi, an expert diagnostic hematopathologist; guidance by physician-scientist advisors; coursework in data science and research ethics; and other professional development activities. All together, I wholeheartedly believe this career development plan will provide a strong foundation on which to ultimately build an independent research program.