Control of ribosomal function in cells of the hematopoietic system

项目概要/摘要 翻译是一个动态过程，具有高度特异性，由包括 mRNA 在内的多种因素赋予 转录序列、翻译机器的组成和细胞环境。一个引人注目的示范 这种特异性是组织特异性的（例如颅面异常、骨髓衰竭），而不是整体性质 由核糖体先天性突变引起的人类核糖体病中观察到的发育缺陷 因素。此外，核糖体病驱动的骨髓衰竭的特点是选择性而非整体性。 mRNA 翻译缺陷。因此，甚至核糖体的组成也会影响 mRNA 的翻译 以组织特异性的方式提高效率。这些观察结果表明了一项特别细致的监管 造血系统的翻译；因此，阐明这一监管机制至关重要 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机制 正常免疫功能和疾病状态下的血淋巴细胞，重点关注核糖体相关 蛋白质（RAP）——一类研究很少的蛋白质，它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。我的中心假设是，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， 它们可能会导致特定 mRNA 翻译效率的快速变化，并具有重要意义。 免疫调节后果。在之前的工作中，我已经定义了核糖体结合 RAP 的全部内容 体外骨髓源性巨噬细胞。在本提案的目标 1 中，我将使用全局、公正的蛋白质组学来 时间分析 TLR 刺激后激活的巨噬细胞中 RAP-核糖体相互作用。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) 识别 mRNA 靶标 受到我已经确定的两个 RAP（Rnf213 和 Helz2）以及其他 RAP 的具体监管 目标 1 和 2 中确定的 RAP 确定了这些 RAP 在巨噬细胞炎症反应中的功能。 完成了医学博士-博士培训以及临床病理学家和血液病理学家的临床培训， 我正在申请 K38 奖，以支持我成为一名独立医师研究者的目标。加州大学旧金山分校 卓越的培训环境，特别是在免疫学和蛋白质稳态领域，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 Davide Ruggero 博士是核糖体生物学领域的先驱，我的共同导师 Davide Ruggero 博士是核糖体生物学领域的先驱。 Jason Cyster 是一位免疫学家专家，Karthik Ganapathi 博士是一位诊断血液病理学家专家； 医师科学家顾问的指导；数据科学和研究伦理课程；以及其他专业 发展活动。总之，我坚信这个职业发展计划将为 最终建立独立研究计划的基础。

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.