5' exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly.
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.
Reichert
Vienna L
VL
Howard Hughes Medical Institute, Department of Biochemistry, Brandeis University, Massachusetts 02454, USA.
Le Hir
Hervé
H
Jurica
Melissa S
MS
Moore
Melissa J
MJ
D005091:Exons / Q000235:genetics*; D006801:Humans; D012322:RNA Precursors / Q000235:genetics*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000235:genetics; D012261:Ribonucleoproteins / Q000235:genetics; D017381:Spliceosomes / Q000235:genetics*
D012322:RNA Precursors; D012333:RNA, Messenger; D012261:Ribonucleoproteins; C026452:messenger ribonucleoprotein