Regulation of Protein Production Dynamics:RNA Binding Proteins and the Ribosome Code

摘要 分子生物学的中心教条假定基因表达是从基因到蛋白质的线性路径。现在是时候了 明确基因表达在几个水平上受到严格控制--从转录到翻译再到蛋白质 降解-然而，分子、细胞和发育生物学领域主要关注转录 控制是基因调控的主要方式。然而，哺乳动物的基因组编码了1500多个RNA结合 蛋白质(RBP)，其中几种在癌症和神经疾病等疾病中反复突变 紊乱，提示转录后基因表达调节，特别是mRNA翻译是 对健康和人类疾病都很重要。此外，到目前为止，核糖体本身的调节作用 没有得到充分的开发。越来越多的证据表明，核糖体蛋白质中的特殊核糖体 化学计量学和翻译后修饰的存在可能会影响特定mRNA的翻译 通过一种尚未定义的“核糖体密码”。 该实验室的主要研究目标是了解翻译的原理和机制 调控控制基因表达的动态，因此影响分化、应激等过程 反应和发病机制。在接下来的五年里，我们将重点关注翻译控制的两个具体方面 在小鼠胚胎干细胞分化的背景下。首先，我们将系统地识别和表征 调节翻译变化的RNA结合蛋白(RBPs)。在我们以前工作的基础上，我们将结合 基于CRISPR的高通量筛选与RNA动态和蛋白质生产的全球测量 和退化。这将把限制性商业惯例与它们的信使核糖核酸靶标联系起来，为未来详细的功能奠定基础。 后续，使我们能够阐明限制性商业惯例如何调控mRNA翻译的功能性和因果洞察力。 其次，我们正在研究核糖体的异质性程度，以检验特化核糖体的假设 核糖体存在选择性地翻译mRNAs的亚群，从而引入额外的调节水平。 基因表达--一种核糖体密码。通过应用高精度质谱学，我们现在正在聚焦 核糖体异质性的两个潜在来源--核心核糖体蛋白的差异表达 以及翻译后修饰的变化。根据这些测量到的核糖体变化 为了进一步进行功能鉴定，我们选择了变化最大的RPS和PTM。 详细的后续行动将提供一套选定的RPS和PTM原则和机械性见解 核糖体专门化如何调控翻译。 这两种方法结合在一起，将提供对蛋白质生产动态的前所未有的洞察 重要的生理背景，潜在地揭开了基因表达调控的新范例。

Summary The central dogma of molecular biology assumes a linear path of gene expression from gene to protein. It is now clear that gene expression is tightly controlled at several levels - from transcription to translation to protein degradation - yet the fields of molecular, cell and developmental biology have mostly focused on transcriptional control as the primary mode of gene regulation. Yet, the mammalian genome encodes over 1,500 RNA binding proteins (RBPs), several of which are recurrently mutated in diseases, such as cancer and neurological disorders, suggesting that post-transcriptional gene expression regulation and especially mRNA translation are important in both health and human disease. Furthermore, the regulatory role of the ribosome itself has so far been under-explored. Evidence is mounting that specialized ribosomes, which vary in ribosomal protein stoichiometry and post-translational modifications, exist that may impact the translation of specific mRNAs through an as-yet-undefined ‘ribosome code’. The overarching research goal of the lab is to understand the principles and mechanisms by which translational regulation controls the dynamics of gene expression and therefore affects processes like differentiation, stress response and pathogenesis. Over the next five years, we will focus on two specific aspects of translational control in the context of mouse embryonic stem cell differentiation. First, we will systematically identify and characterize RNA binding proteins (RBPs) that regulate translational changes. Based on our previous work, we will combine high-throughput CRISPR-based screening with global measurements of RNA dynamics, and protein production and degradation. This will link RBPs to their mRNA targets, providing the foundation for future detailed functional follow-ups, allowing us to elucidate functional and causal insights of how RBPs regulate mRNA translation. Second, we are looking at the extent of ribosomal heterogeneity, testing the hypothesis that specialized ribosomes exist that selectively translate subsets of mRNAs, thereby introducing an additional level of regulation in gene expression – a ribosome code. By applying high accuracy mass spectrometry, we are focusing right now on two potential sources of ribosomal heterogeneity – differential expression in core ribosomal proteins (RPs) and changes in their post-translational modifications. Based on these measured changes in ribosome composition, we are selecting RPs and PTMs with the strongest changes for further functional characterization. The detailed follow up will provide for a selected set of RPs and PTMs the principles and mechanistic insight how ribosome specialization regulates translation. Together these two approaches will provide unprecedented insight on the dynamics of protein production in an important physiological context, potentially unravelling novel paradigms of gene expression regulation.