Structural investigation of protein splicing factors that promote the formation of the spliceosomes catalytic centre

前mRNA剪接发生在两个化学步骤中，由称为剪接体的大型动态RNA-蛋白质复合物催化。剪接体最初以无催化活性的形式组装，经历大规模的组成和构象重塑，通过这些重塑，远程RNA元件被重新配置成功能性催化中心。这个复杂的过程-催化中心的形成-需要大量蛋白质的补充。其中最重要的是蛋白质Cwc 2以及预组装成所谓的十九复合物（NTC）的八种蛋白质。目前的数据表明，NTC，剪接体的主要组成部分之一，在剪接周期的各个阶段执行几项功能任务。尽管到目前为止进行了许多生物化学和遗传学研究，但NTC功能的综合图像仍然难以捉摸，主要是由于缺乏原子分辨率的3D模型。因此，我们的主要目标之一是重建的NTC和确定其分子内结构的X射线晶体学。鉴于这种550-kDa复合物的结晶困难，我们首先将目光放在NTC亚复合物NTCPrp 19和NTCSyf 1的结晶上。虽然NTC对RNA催化中心的形成至关重要，但NTC和RNA之间的唯一物理连接是由Cwc 2介导的，Cwc 2是一种与RNA催化中心直接接触的蛋白质。由于Cwc 2-NTC相互作用可能解释NTC如何间接诱导催化中心的功能构象，我们的目的是研究原子细节的NTC-Cwc 2关系。这些蛋白质和蛋白质复合物的晶体结构将通过各种生物化学和生物物理学研究来补充，以获得对整个剪接体功能的更深层次的机理见解。

Pre-mRNA splicing occurs in two chemical steps that are catalysed by a large, dynamic RNA-protein complex called the spliceosome. Initially assembled in a catalytically inactive form, the spliceosome undergoes massive compositional and conformational remodelling, through which remote RNA elements are re-configured into a functional catalytic centre. This intricate process- the formation of the catalytic centre - requires the recruitment of numerous proteins. Among the most important of these is the protein Cwc2 as well as eight proteins that are pre-assembled into the so-called nineteen complex (NTC). Current data suggest that the NTC, one of the major building blocks of the spliceosome, performs several functional tasks at various stages of the splicing cycle. Despite the many biochemical and genetic studies conducted so far, an integrated picture of the function of the NTC remains elusive, mainly owing to the absence of a 3D model at atomic resolution. One of our major goals is therefore the reconstitution of the NTC and the determination of its intramolecular architecture by X-ray crystallography. In view of the difficulty of crystallising this 550-kDa complex, we would first set our sights on the crystallisation of the NTC subcomplexes NTCPrp19 and NTCSyf1.Although the NTC is crucial for the formation of the RNA-based catalytic centre, the only physical connection between the NTC and RNA is mediated by Cwc2, a protein that makes direct contact with the RNA-based catalytic centre. As the Cwc2-NTC interaction might explain how the NTC indirectly induces a functional conformation of the catalytic centre, our aim is to investigate the NTC-Cwc2 relation in atomic detail. The crystal structures of these proteins and protein complexes will be complemented by various biochemical and biophysical studies, in order to obtain deeper mechanistic insight into the function of the entire spliceosome.