Structural investigation of Aquarius and associated splicing factors that couple pre-mRNA splicing to downstream RNA-processing events

剪接体是一种核糖核蛋白机器，它组装在mRNA前体上，催化内含子的切除和外显子的连接。整个剪接途径是高度动态的，剪接体在RNA解旋酶的运动活性驱动下经历了许多重塑步骤。除了参与剪接本身，一些剪接体成分在剪接与其他RNA代谢事件的偶联中起关键作用。Aquarius是一种巨大的剪接体解旋酶，它将内含子结合在一个精确的位置，并将剪接与mRNA上外显子连接复合物的沉积以及内含子片段的snornp形成结合。水瓶座的结构在很大程度上是未知的，它用于耦合的机制也不被理解。越来越多的证据表明，Aquarius作为一个五聚体复合体的一部分被招募到剪接体上，我们称之为IBC（内含子结合复合体）。随后，Aquarius和其他IBC成分稳定地整合到剪接体中，直到剪接途径的最后阶段。阐明Aquarius的机制作用，以及隐含的IBC在剪接体重塑与下游rna加工事件耦合中的作用，将通过它们在原子分辨率上的3D表征得到极大的推进。因此，我们的主要目标是水瓶座的结晶和X射线结构的确定，它与RNA的复合物和整个IBC。晶体学研究将辅以生物化学和生物物理分析，在孤立和整个剪接体的背景下。

The spliceosome is a ribonucleoprotein machine that assembles on the pre mRNA and there catalyses the excision of introns and the ligation of exons. The entire splicing pathway is highly dynamic, with the spliceosome undergoing numerous remodelling steps driven by the motor activity of RNA helicases. Apart from participating in splicing per se, some of the spliceosomal components play pivotal roles in coupling splicing to other events of RNA metabolism.Aquarius is a massive spliceosomal helicase which binds the intron at a precise location and couples splicing to the deposition of the exon-junction complex on the mRNA as well as to the formation of snoRNPs from intronic segments. The structure of Aquarius is largely unknown and the mechanism that it employs for coupling is not understood.Converging evidence suggests that Aquarius is recruited to the spliceosome as part of a pentameric complex that we denote IBC (intron-binding complex). Subsequently, Aquarius and the other IBC components remain stably integrated into the spliceosome until the final stages of the splicing pathway. Elucidating the mechanistic role of Aquarius and, implicitly, that of the IBC in coupling spliceosomal remodelling to the downstream RNA-processing events would be greatly advanced by their 3D characterisation at atomic resolution. Therefore, our main goals are the crystallisation and X ray structure determination of Aquarius, of its complex with RNA and of the entire IBC. The crystallographic studies will be complemented with biochemical and biophysical analyses, in isolation and in the context of the whole spliceosome.