The RNA-bound proteome

RNA和蛋白质之间的功能相互作用是基因表达的基础，而RNA-蛋白质相互作用中的缺陷构成了许多人类疾病的基础。RNA结合蛋白在基因表达的各个环节都具有重要的功能，包括转录、RNA加工、核输出和mRNA翻译，以及病毒防御机制。David Tollervey是一位RNA生物学专家，他的团队最近开发了总RNA相关蛋白纯化(TRapp)的方法(参考文献2)。1)。这是基于体内蛋白质-RNA复合体的UV交联和变性纯化，结合使用SILAC代谢标记的定量。TRapp可以在蛋白质组范围内快速鉴定RNA结合蛋白，并对营养物质转移、应激或发育过程中的变化进行量化。这项技术依靠串联MS-MS来产生类似于鉴定其他蛋白质修饰位点的片段。相关的iTRAPP技术允许通过氨基酸解析来绘制蛋白质-RNA相互作用的精确位置图。值得注意的是，iTRAPP可用于表征内在无序区域内的RNA-蛋白质相互作用，这是许多RNA结合蛋白质的重要特征，但对于传统的结构分析来说在很大程度上是困难的(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，他的团队开发了重要的质谱学技术来表征蛋白质组变化的动态。？？)。该项目旨在应用这些方法来跟踪在生物学上重要的转变期间RNA-蛋白质相互作用的变化。为了进一步发展动力学分析，我们首先跟踪了酵母中的应激反应。我们最近的工作发现，核RNA降解是基因表达中一个积极调节的步骤。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新编程的情况下发挥重要作用，例如后生动物的发育步骤。交互作用组的变化和所涉及的信号通路将使用TRapp来识别。被鉴定为可能具有功能重要性的蛋白质的RNA相互作用的具体变化将通过交联和RNA测序来表征(见参考文献。2)。这也将在博士项目期间进行。随后，学生将把从酵母分析中获得的见解应用到使用人类细胞的研究中。RNA结合蛋白的缺陷是许多遗传性疾病的基础，但在大多数情况下，特定的分子缺陷与受影响细胞的系统效应之间的联系尚不清楚。这将使用TRapp来解决，特别强调神经元发育模型系统中在分化过程中发生的变化。所涉及的工作：学生将进一步开发和应用描述和量化RNA-蛋白质相互作用变化的技术，并分析所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方面的专业知识。

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.