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

促进剪接体催化中心形成的蛋白质剪接因子的结构研究

• 批准号: 227670291
• 负责人: Dr. Vladimir Pena, Ph.D.
• 金额: --
• 依托单位: Institute of Cancer Research
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2013
• 资助国家: 德国
• 起止时间: 2012-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/227670291?language=en
• 关键词: Structural; investigation; protein; splicing; factors

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.

mRNA 前体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。剪接体最初以催化非活性形式组装，经历大规模的组成和构象重塑，通过这种重塑，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——需要招募大量蛋白质。其中最重要的是蛋白质 Cwc2 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 是剪接体的主要构建模块之一，在剪接周期的不同阶段执行多种功能任务。尽管迄今为止进行了许多生化和遗传学研究，但 NTC 功能的完整图像仍然难以捉摸，这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 NTC 并通过 X 射线晶体学测定其分子内结构。鉴于结晶这个 550 kDa 复合物的难度，我们首先将目光投向 NTC 子复合物 NTCPrp19 和 NTCSyf1 的结晶。虽然 NTC 对于基于 RNA 的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 Cwc2 介导的，Cwc2 是一种与基于 RNA 的催化中心直接接触的蛋白质。 中心。由于 Cwc2-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，因此我们的目标是在原子细节上研究 NTC-Cwc2 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化和生物物理研究得到补充，以获得对整个剪接体功能的更深入的机制了解。