5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

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Abstract
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.