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

总结 分子生物学的中心法则假设基因表达从基因到蛋白质的线性路径。现在 很明显，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 然而，分子、细胞和发育生物学领域主要集中在转录水平上， 控制是基因调控的主要方式。然而，哺乳动物基因组编码超过1,500种RNA结合， 蛋白质（RBP），其中几种在疾病中反复突变，如癌症和神经系统疾病。 疾病，这表明转录后基因表达调控，特别是mRNA翻译， 对健康和人类疾病都很重要。此外，迄今为止，核糖体本身的调节作用 被低估了。越来越多的证据表明，特殊的核糖体，在核糖体蛋白质的变化， 化学计量和翻译后修饰，存在可能影响特定mRNA的翻译 通过一个尚未定义的“核糖体密码”。 该实验室的首要研究目标是了解翻译的原则和机制， 调控控制着基因表达的动力学，因此影响着分化、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描述 RNA结合蛋白（RBP），调节翻译变化。基于我们以前的工作，我们将联合收割机 基于CRISPR的高通量筛选，全面测量RNA动力学和蛋白质生产 和退化。这将把RBP与它们的mRNA靶点联系起来，为将来详细的功能性研究提供基础。 随访，使我们能够阐明RBP如何调节mRNA翻译的功能和因果关系的见解。 第二，我们正在研究核糖体异质性的程度，检验特化核糖体的假说。 核糖体的存在，选择性地翻译mRNA的子集，从而引入了额外的调节水平 在基因表达-核糖体密码。通过应用高精度质谱仪，我们现在正在关注 核糖体异质性的两个潜在来源-核心核糖体蛋白（RP）的差异表达 以及它们的翻译后修饰的变化。基于这些测量到的核糖体变化 为了进一步确定组成，我们选择具有最强变化的RP和PTM用于进一步的功能表征。 详细的后续行动将为一组选定的RP和PTM提供原则和机制见解 核糖体特化如何调节翻译。 这两种方法将共同提供对蛋白质生产动态的前所未有的洞察力， 重要的生理背景，潜在地解开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.