Studies of multi-component complexes by NMR: application to viral mRNA export

包括人类在内的高等生物的细胞以DNA的形式将遗传信息存储在称为细胞核的中央隔室中。在一个称为转录的过程中，DNA的选定部分被读取，信息随后被编码成信使RNA（mRNA）分子。这些分子经过进一步加工和重排（剪接），从而为合成各种不同的蛋白质提供了蓝图。这些蓝图从细胞核传递到细胞的其他部分，在那里细胞机器使用它们来生产蛋白质。信使RNA的加工、重排和从细胞核输出是由剪接和输出机制的各种蛋白质组分主动进行的。这些成分与信使RNA相互作用，并相互作用，施加控制，使只有成熟的细胞mRNA被输出。这个控制系统是如何工作的还不完全清楚。一些病毒产生的蛋白质利用这种细胞输出机制，欺骗它从细胞核携带病毒遗传信息，绕过细胞控制机制。这样，病毒以细胞为代价维持自己的生命周期;这到底是如何发生的还需要进一步研究。要更好地了解细胞mRNA输出系统的功能（或病毒感染期间内部控制机制如何绕过），需要表征其单个蛋白质组分的3D结构，动态和结合特性，并研究它们在相互结合时的行为。拟议的研究旨在研究病毒蛋白片段与负责mRNA输出的关键天然细胞蛋白之间相互作用的分子基础，这一领域与我们最近的成功研究密切相关。我们的初步结果表明，在多组分分子复合物中，病毒mRNA可以从一个蛋白质分子转移到另一个蛋白质分子：如何实现这一点将在拟议的研究中揭示。申请人实验室开创的新技术将用于详细研究系统中不同的多个分子组分如何相互作用，以及不同的分子区域如何在该过程中发挥作用。揭示病毒蛋白质劫持细胞mRNA输出系统的结构机制可能会导致新的抗病毒药物的开发。该项目的方法方面也具有更广泛的影响。一般来说，蛋白质-蛋白质相互作用在生物过程的几乎所有方面都起着关键作用。在细胞内，许多蛋白质参与多蛋白质复合物，无论是瞬时或稳定，导致复杂的蛋白质相互作用网络。在多蛋白复合物中结合和解离事件的精确排序背后的分子机制仍然不清楚，通常难以研究，但提供了一个非常感兴趣和重要的主题。未来几代特异性药物可能会靶向关键的蛋白质-蛋白质相互作用，诱导或抑制它们。所有这些反过来又提高了对新方法的兴趣，通过该方法来分析在样品管中重建的复杂的多蛋白质相互作用，以及创建可以检测、探测和表征这种相互作用的测定。本项目提出的研究复杂相互作用的新策略解决了这一问题，并将适用于其他多蛋白质相互作用系统，并有望推动整个蛋白质相互作用研究领域的研究，包括纳米技术和药物设计。

The cells of higher organisms including humans store the genetic information in a form of DNA in a central compartment known as nucleus. In a process called transcription selected parts of DNA are read and information is subsequently coded into messenger RNA (mRNA) molecules. These molecules are subject to further processing and re-arrangements (splicing), consequently providing a blueprint for synthesis of an enormous variety of different proteins. These blueprints are delivered from the nucleus to other parts of the cell, where the cell machinery uses them to produce proteins. Messenger RNA processing, re-arrangement and export from the nucleus are actively performed by various protein components of the splicing and export machinery. These components interact with messenger RNAs and with each other, imposing control so that only mature cellular mRNA is exported. How this control system works is not completely clear. Some viruses produce proteins which make use of this cellular export machinery, tricking it into carrying viral genetic information from the nucleus, and by-passing cellular control mechanisms. In this way the viruses support their own life cycle at the cell's expense; how exactly this happens still needs further research. Better understanding of how the cellular mRNA export system functions (or how internal control mechanisms are by-passed during viral infection) requires characterisation of 3D structural, dynamic and binding properties of its individual protein components, and investigation of how they behave when binding with each other. The proposed research aims at studying molecular basis of interactions between fragments of viral proteins and key native cellular proteins responsible for mRNA export, an area immediately related to our recent successful research. Our preliminary results show that in multi-component molecular complex the viral mRNA may be transferred from one protein molecule to another: how this is achieved will be revealed in the proposed study. The new technique pioneered in the applicant's laboratory will be used to look in detail at how different multiple molecular components of the system interact together, and how different molecular regions operate in the process. Revealing the structural mechanism by which viral proteins hijack the cellular mRNA export system potentially can lead to development of new anti-viral drugs. The methodological aspect of the project also has wider implications. In general, protein-protein interactions play a critical role in virtually all aspects of biological processes. Within a cell, many proteins participate in multi-protein complexes, either transiently or stably, resulting in complex protein interaction networks. The molecular mechanisms behind the precise ordering of binding and dissociation events in multi-protein complexes remain unclear and are generally difficult to study, but provide a topic of utmost interest and importance. The future generations of specific drugs are likely to target key protein-protein interactions, either inducing or suppressing them. All this in turn boosts the interest in new methods by which to analyze complex multi-protein interactions reconstructed in sample tube, and in creating assays where such interactions can be detected, probed and characterized. The novel strategy of investigation of complex interactions proposed in the current project addresses this issue and will be applicable to other multi-protein interaction systems, and is expected to boost the research in the whole field of protein interactions studies, including nanotechnology and drug design.