Mapping global mRNA fate: integrating translational and spatial dynamics

活细胞中基因的信息内容被解码，产生被称为蛋白质的不同氨基酸链，这些氨基酸链决定了细胞的身份和功能。蛋白质是生物功能的主要效应器，负责催化大多数生化反应，以及服务于许多结构和调节角色。它们从中间分子信使RNA （mRNA）翻译成蛋白质，整个过程在所有真核细胞（动物、植物和真菌）中都非常相似。越来越清楚的是，这种翻译过程是控制蛋白质水平（从而控制生物功能和细胞状态）的关键调控步骤，既通过改变特定mrna的翻译水平，也通过靶向位置。蛋白质和mrna都可以在细胞内定位，以促进特定蛋白质的局部浓度的产生，这在特定细胞区域（如神经元轴突或肠细胞微绒毛）的空间发育中起着关键作用。mRNA定位到细胞中的特定位点通常涉及颗粒，颗粒中含有惰性的mRNA，翻译抑制状态。mrna也可以在细胞逆境中被定位，其中已经鉴定出两种不同类型的颗粒，“应激颗粒”和“p体”。这些颗粒被认为在储存有用的mrna和破坏多余的mrna中发挥作用。此外，它们的分布还与人类疾病，特别是大脑和肌肉疾病有关，并在多细胞动物的发育，特别是胚胎的发育中起着根本作用。由于mRNA在特定细胞区域的定位已经在酵母中得到了广泛的研究，将细胞应激与p小体和应激颗粒中的mRNA定位联系起来，我们的目标是利用这个简单的模式生物来揭示这一过程的基本分子生物学，以控制蛋白质合成。我们最近的研究发现了两个新发现。令人惊讶的是，即使在活跃生长的细胞中，编码非定位蛋白质的mRNA也存在于mRNA颗粒中，这些蛋白质参与糖代谢和翻译本身等常规途径。我们的实验表明mRNA翻译成蛋白质发生在这些颗粒中。在第二项研究中，我们发现大多数这些定位mrna在蛋白质合成选择的经典“闭环”模型中不相互作用，因此不清楚这些mrna是如何翻译的。在这个项目中，我们将使用尖端的分子技术来破译颗粒中存在哪些mrna和蛋白质，找出它们是如何翻译的，并探索它们定位的生物学原因。我们将研究mrna是如何被定位的，并评估它们是如何传递给子细胞的。具体来说，我们将确定来自相同途径或复合物的蛋白质是否在这些位点协同产生和调节。我们还将验证关键转录本通过这些颗粒传递给子细胞的假设，作为为后代发育提供关键mrna“启动包”的一种手段。虽然酵母是一种简单的真核生物，但所有已知的酵母翻译控制机制都存在于哺乳动物细胞中。因此，我们对酵母的基础研究将指导和指导包括人类在内的其他系统的研究，并为酵母常用的工业生物技术表达系统提供替代机制。本提案中的研究可能很好地允许在这个水平上进行优化，特别是对于多蛋白复合物。

The information content of genes in living cells is decoded to produce chains of different amino acids called proteins that dictate the identity and function of a cell. Proteins are the principal effectors of biological function, responsible for catalysing most biochemical reactions, as well as serving numerous structural and regulatory roles. They are translated into protein from an intermediate molecule, messenger RNA (mRNA), by a process that is highly similar across all eukaryotic cells (animals, plants and fungi). It is becoming increasingly clear that this translation process is a key regulatory step in the control of protein level (and hence biological function and cellular state), both by changing the level of translation of specific mRNAs and also by targeting the location. Both proteins and mRNAs can be localised within cells to facilitate the generation of local concentrations of particular proteins, and this plays critical roles in the spatial development of specific cellular zones such as axons on neurons or microvilli on intestinal cells. mRNA localisation to specific sites in cells usually involves granules which contain the mRNAs in an inert, translationally repressed state. mRNAs can also become localised during times of cellular adversity, where two different classes of granule have been identified, 'stress granules' and 'P-bodies'. These granules are thought to play roles in both the storage of useful and destruction of surplus mRNAs. Additionally, their deployment has also been linked to human disease, especially in diseases of the brain and muscles, as well as fundamental roles in the development of multicellular animals, especially development of the embryo. Since mRNA localisation to defined cellular regions has been widely studied in yeast, linking cellular stress to mRNA localisation in both P-bodies and stress granules, we aim to use this simple model organism to uncover the fundamental molecular biology of this process for the control of protein synthesis. Our recent studies have uncovered two novel findings. Surprisingly, mRNAs encoding unlocalised proteins involved in routine pathways such as sugar metabolism and translation itself are present in mRNA granules, even in actively growing cells. Our experiments suggest that mRNA translation into protein occurs in these granules. In a second study, we have found that most of these localised mRNAs do not interact in a classical "closed loop" model of selection for protein synthesis, making it unclear how these mRNAs are translated. In this project we will use cutting-edge molecular technologies to decipher which mRNAs and proteins are present in the granules, find out how they are translated, and explore the biological reasons for their localisation. We will examine how mRNAs become localised and also assess how they are passed on to their daughter cells. Specifically, we will determine whether proteins from the same pathway or complex are co-ordinately produced and regulated at these sites. We will also test the hypothesis that key transcripts are passed on to daughter cells via these granules, as a means to provide a "start-up pack" of key mRNAs for developing progeny.Although yeast is a simple eukaryote, all known mechanisms of translational control utilised in yeast are present in mammalian cells. Hence, our fundamental studies in yeast will guide and inform studies in other systems including human, as well as provide alternative mechanisms to tweak industrial biotechnology expression systems where yeast is commonly used. The studies in this proposal may well allow optimisation at this level, especially for multi-protein complexes.