Dissecting ribosome pausing during embryogenesis: from global and single molecule studies to whole embryo phenotypes

生物体的基因是蛋白质产生的蓝图，然而在任何给定的单个细胞中，只有所有基因的一个子集最终被解码以产生蛋白质。因此，细胞中蛋白质的特性和丰度决定了它是否会成为大脑中的神经细胞或肠内膜中的上皮细胞。这在生物体的发育过程中尤为显著，在不同的生长阶段，不同的蛋白质组被制造出来，从而形成不同的细胞类型。蛋白质是构成生命基石的氨基酸链。蛋白质中氨基酸的顺序和身份是在称为“翻译”的过程中从称为mRNA的基因中间拷贝解码的。核糖体是细胞内负责翻译的机器。它们与mRNA结合，并根据从mRNA读取的代码依次将氨基酸插入生长的蛋白质链上。然而，并非所有的氨基酸插入事件都以相同的效率发生，并且由于许多原因，核糖体在mRNA的粘片上暂停。在各种生物学背景下，这些核糖体停顿是重要的。然而，由于在mRNA单分子上跟踪核糖体的技术挑战，只有少数核糖体暂停事件得到了非常详细的研究。我们的研究使用经典的模型来研究核糖体暂停，果蝇，因为从受精卵到成年果蝇的发育只需要十天，这代表了一个奇妙的系统，可以研究蛋白质生产的变化如何影响复杂的多细胞动物的发育。甚至在果蝇胚胎的早期阶段，成体的基本分节模式就变得明显了。因此，胚胎内特定部位关键蛋白质的精确定时生产决定了哪些组织将发育，以及它们将在何时何地发育。在这个提议中，我们的目标是确定核糖体暂停如何控制蛋白质生产的时间，以便在胚胎发育过程中形成正确的细胞类型。为了实现这一目标，我们将使用最先进的显微镜和测序方法，这将使我们能够回答三个关键问题。首先，哪些mrna暂停了果蝇胚胎中的核糖体？其次，是什么让这些mrna让核糖体暂停？第三，破坏核糖体暂停如何影响果蝇胚胎的发育？总的来说，我们的数据将为指导核糖体暂停的信号和胚胎发育过程中受影响的过程范围提供重要的新信息。由于所有已知的翻译控制机制在动物和植物细胞中都是保守的，因此本研究的结果将与人类发育直接相关。因此，我们的发现对于理解许多与翻译伸长调控不当相关的人类疾病将是重要的。最后，我们的数据也将有利于干细胞研究，在干细胞研究中，操纵基因表达以有效地将干细胞分化为特定细胞类型的能力是一个主要的治疗目标。

An organism's genes are the blueprint from which proteins are produced, yet in any given single cell, only a subset of all genes is ultimately decoded to give proteins. As such, the identity and abundance of proteins made in a cell determines whether it will become, say a nerve cell in the brain or an epithelial cell in the intestinal lining. This is particularly striking during the development of an organism, where distinct sets of proteins are made at various stages in growth so that different cell types form. Proteins are chains of amino acids which form the building blocks of life. The order and identity of amino acids in a protein is decoded from an intermediate copy of genes called a mRNA in a process termed 'translation'. Ribosomes are the machines inside cells responsible for translation. They bind to the mRNA and sequentially insert amino acids onto a growing protein chain according to the code read from the mRNA. However, not all amino acid insertion events occur with the same efficiency and for many reasons ribosomes pause at sticky patches on a mRNA. In various biological contexts these ribosome pauses are important. However, only a handful of ribosome pausing events have been studied in great detail owing to the technical challenges of following ribosomes on single molecules of mRNA.Our research studies ribosome pausing using the classic model, the fruitfly Drosophila, since the development of an adult fruitfly from a fertilised egg only takes ten days and represents a wonderful system to study how changes in protein production affect the development of a complex multicellular animal. Even at the earliest stages in the fruitfly embryo, the basic segmented pattern of the adult body is becoming apparent. Therefore, the precisely timed production of key proteins at specific sites within the embryo dictates which tissues will develop, and when/ where they will do so. In this proposal, we aim to determine how ribosome pausing controls the timing of protein production so that the correct cell types form during embryonic development. To achieve this goal, we will use state-of-the-art microscopy and sequencing approaches, which will allow us to answer three key questions. Firstly, which mRNAs have paused ribosomes in the fruitfly embryo? Secondly, what is it about these mRNAs that makes the ribosomes pause? Thirdly, how does disrupting ribosome pausing affect development of the fruitfly embryo?Overall, our data will provide important new information about the signals that direct ribosome pausing and the range of processes affected during embryonic development. As all known mechanisms of translational control are conserved across animal and plant cells, results from this study will be directly relevant to human development. Therefore, our findings will be important for understanding the many human diseases that are associated with misregulation of translation elongation. Finally, our data will also benefit stem cell research, where the ability to manipulate gene expression to efficiently differentiate stem cells into particular cell types is a major therapeutic goal.