Reichert Vienna L, Le Hir Hervé, Jurica Melissa S, Moore Melissa J
Howard Hughes Medical Institute, Department of Biochemistry, Brandeis University, Massachusetts 02454, USA.
Genes Dev. 2002 Nov 1;16(21):2778-91. doi: 10.1101/gad.1030602.
A general consequence of pre-mRNA splicing is the stable deposition of several proteins 20-24 nucleotides (nt) upstream of exon-exon junctions on spliced mRNAs. This exon junction complex (EJC) contains factors involved in mRNA export, cytoplasmic localization, and nonsense-mediated mRNA decay. Here we probed the mechanism and timing of EJC assembly. Over the course of splicing, the 5' exon is subject to numerous dynamic protein-RNA interactions involving at least nine distinct polypeptides. Within the fully assembled spliceosome, these interactions afford protection to the last 25-27 nt of the 5' exon intermediate. Coincident with exon ligation, interactions at the 3' end of the 5' exon disappear, and new species associate with position -24. Mass spectrometry and Western blotting of purified H, C, and mRNP complexes revealed that at least one EJC component, REF/Aly, can interact with pre-mRNA prior to spliceosome assembly, whereas Y14, Magoh, RNPS1, UAP56, and SRm160 are found in intermediate-containing spliceosomes. Upon exon ligation, association of RNPS1, UAP56, and SRm160 is destabilized. In contrast, REF/Aly, Y14, and Magoh remain stably bound to spliced mRNA, indicating that these three proteins are components of the EJC core.
前体mRNA剪接的一个普遍结果是，几种蛋白质稳定沉积在剪接后mRNA外显子-外显子连接点上游20 - 24个核苷酸（nt）处。这种外显子连接复合体（EJC）包含参与mRNA输出、细胞质定位和无义介导的mRNA降解的因子。在这里，我们探究了EJC组装的机制和时机。在剪接过程中，5'外显子会经历大量动态的蛋白质-RNA相互作用，涉及至少九种不同的多肽。在完全组装的剪接体中，这些相互作用为5'外显子中间体的最后25 - 27 nt提供保护。与外显子连接同时发生的是，5'外显子3'端的相互作用消失，新的蛋白种类与位置-24相关联。对纯化的H、C和mRNP复合体进行质谱分析和蛋白质免疫印迹分析表明，至少一种EJC组分REF/Aly在剪接体组装之前就能与前体mRNA相互作用，而Y14、Magoh、RNPS1、UAP56和SRm160存在于含有中间体的剪接体中。外显子连接后，RNPS1、UAP56和SRm160的结合变得不稳定。相反，REF/Aly、Y14和Magoh仍稳定地结合在剪接后的mRNA上，表明这三种蛋白质是EJC核心的组分。
