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首页> 外文期刊>Annual review of biophysics >Cryo-EM Studies of Pre-mRNA Splicing: From Sample Preparation to Model Visualization
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Cryo-EM Studies of Pre-mRNA Splicing: From Sample Preparation to Model Visualization

机译：前mRNA拼接的Cryo-EM研究：从样品制备到模型可视化

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摘要

The removal of noncoding introns from pre-messenger RNA(pre-mRNA) is an essential step in eukaryotic gene expression and is catalyzed by a dynamic multi-megadalton ribonucleoprotein complex called the spliceosome. The spliceosome assembles on pre-mRNA substrates by the stepwise addition of small nuclear ribonucleoprotein particles and numerous protein factors. Extensive remodeling is required to form the RNA-based active site and to mediate the pre-mRNA branching and ligation reactions. In the past two years, cryo-electron microscopy (cryo-EM) structures of spliceosomes captured in different assembly and catalytic states have greatly advanced our understanding of its mechanism. This was made possible by long-standing efforts in the purification of spliceosome intermediates as well as recent developments in cryo-EM imaging and computational methodology. The resulting high-resolution densities allow for de novo model building in core regions of the complexes. In peripheral and less ordered regions, the combination of cross-linking, bioinformatics, biochemical, and genetic data is essential for accurate modeling. Here, we summarize these achievements and highlight the critical steps in obtaining near-atomic resolution structures of the spliceosome.
机译：从前信使RNA（前mRNA）的去除非编码内含子是真核基因表达中的基本步骤，并通过称为抗缩丝体的动态多百百醛核糖核糖蛋白络合物催化。通过逐步添加小核核糖核蛋白颗粒和许多蛋白质因子，抗乳头组在前mRNA底物上组合。需要进行广泛的重塑以形成基于RNA的活性位点，并介导前mRNA分支和连接反应。在过去两年中，在不同组装和催化状态中捕获的抗滑微观物（Cryo-Em）结构具有极大地推进了我们对其机制的理解。这是通过长期努力在纯化抗磷酸体中间体以及Cryo-EM成像和计算方法的最新发展中的长期努力实现的。由此产生的高分辨率密度允许在复合物的核心区域中进行De Novo模型建筑。在外围和较少的订购区域中，交联，生物信息学，生物化学和遗传数据的组合对于精确建模至关重要。在这里，我们总结了这些成就，并突出了获得近似原子分辨率结构的关键步骤。

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