The RNA-bound proteome

RNA和蛋白质之间的功能性相互作用支撑基因表达，并且RNA-蛋白质相互作用中的缺陷形成许多人类疾病的基础。RNA结合蛋白在基因表达的所有步骤中具有重要功能，包括转录、RNA加工、核输出和mRNA翻译，以及在病毒防御机制中。大卫Tollervey是一个专家在RNA生物学和他的小组最近开发的方法总RNA相关蛋白纯化（TRAPP）（参考文献1）。这是基于蛋白质-RNA复合物的体内UV交联和变性纯化，结合使用SILAC代谢标记的定量。TRAPP允许快速，蛋白质组范围内的RNA结合蛋白的鉴定和定量的变化条件下的营养转移，压力或发育进程。该技术依赖于串联MS-MS来产生类似于蛋白质修饰的其他位点的鉴定的片段。相关的iTRAPP技术允许蛋白质-RNA相互作用的精确位点与氨基酸分辨率的映射。值得注意的是，iTRAPP可用于研究内在无序区域内的RNA-蛋白质相互作用，这是许多RNA结合蛋白质的重要特征，但在很大程度上难以进行常规结构分析（见参考文献2）。安格斯拉蒙德是定量蛋白质组学的世界领导者，他的小组已经开发出重要的质谱技术来研究蛋白质组中改变的动力学（参考文献？？）。该项目旨在应用这些方法来跟踪生物学重要转变期间RNA-蛋白质相互作用的变化。为了进一步发展动力学分析，我们首先在酵母中跟踪应激反应。我们最近的工作确定核RNA降解是基因表达中的一个积极调控步骤。我们预计，核RNA降解途径的变化将在其他需要大规模基因表达重编程的情况下发挥重要作用，如后生动物的发育步骤。将使用TRAPP鉴定相互作用组的变化和所涉及的信号通路。将通过交联和RNA测序来表征被鉴定为可能具有功能重要性的蛋白质的RNA相互作用的特定变化（参见参考文献2）。这也将在博士项目期间进行。随后，学生将从分析酵母中获得的见解应用于使用人类细胞的研究。RNA结合蛋白的缺陷是许多遗传疾病的基础，但在大多数情况下，特定分子缺陷与受影响细胞的全身效应之间的联系仍不清楚。这将使用TRAPP解决，特别强调在神经元发育的模型系统中分化过程中发生的变化。涉及的工作：学生将进一步开发和应用RNA-蛋白质相互作用变化的表征和定量技术，并分析由此产生的蛋白质组学和序列数据。在这个项目中，学生将获得广泛的尖端技术的专业知识。

Functional interactions between RNAs and proteins underpin gene expression and defects in RNA-protein interactions form the basis of numerous human diseases. RNA binding proteins have important functions at all steps in gene expression, including transcription, RNA processing, nuclear export and mRNA translation, as well as in viral defence mechanisms. David Tollervey is an expert in RNA biology and his group recently developed the approach of total RNA-associated protein purification (TRAPP) (ref. 1). This is based on in vivo UV crosslinking of protein-RNA complexes and denaturing purification, combined with quantitation using SILAC metabolic labelling. TRAPP allows the rapid, proteome-wide identification of RNA-binding proteins and quantitation of changes under conditions of nutrient shift, stress or developmental progression. This technique relies on tandem MS-MS to generate fragments similar to the identification of other sites of protein modification. The related iTRAPP technique allows the mapping of precise sites of protein-RNA interaction with amino acid resolution. Significantly, iTRAPP can be used to characterise RNA-protein interactions within intrinsically disordered regions, which important features of many RNA-binding proteins but largely intractable for conventional structural analyses (see ref. 2).Angus Lamond is world leader in quantitative proteomics and his group has developed important mass spectrometry techniques to characterise the dynamics of alterations in the proteome (ref. ??).The project aims to apply these approaches to follow changes in RNA-protein interactions during biologically important transitions. To further develop kinetic analyses, we are initially following stress responses in yeast. Our recent work identified nuclear RNA degradation as an actively regulated step in gene expression. We anticipate that changes in nuclear RNA degradation pathways will play important roles in other situations that require large scale reprogramming of gene expression, such as developmental steps in metazoans. Interactome changes and the signalling pathways involved will be identified using TRAPP. Specific changes in the RNA interactions of proteins identified as being of likely functional importance will be characterised by crosslinking and RNA sequencing (see ref. 2). This will also be pursued during the PhD project. Subsequently, the student will apply the insights gained from analyses yeast to studies using human cells. Defects in RNA-binding proteins underpin a large number of genetic diseases, but in most cases the links between the specific molecular defect and the systemic effects in affected cells remain unclear. This will be addressed using TRAPP, with particular emphasis on changes that occur during differentiation in a model system for neuronal development.Work involved: The student will further develop and apply, techniques for the characterisation and quantitation of changes in RNA-protein interactions, and analyse the resulting proteomic and sequence data. During this project the student will acquire expertise in a wide range of cutting-edge techniques.