The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

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Abstract
Newly spliced mRNAs in mammalian cells are characterized by a complex of proteins at exon–exon junctions. This complex recruits Upf3 and Upf2, which function in nonsense‐mediated mRNA decay (NMD). Both Upf proteins are detected on mRNA bound by the major nuclear cap‐binding proteins CBP80/CBP20 but not mRNA bound by the major cytoplasmic cap‐binding protein eIF4E. These and other data indicate that NMD targets CBP80‐bound mRNA during a ‘pioneer’ round of translation, but whether nuclear eIF4E also binds nascent but dead‐end transcripts is unclear. Here we provide evidence that nuclear CBP80 but not nuclear eIF4E is readily detected in association with intron‐containing RNA and the C‐terminal domain of RNA polymerase II. Consistent with this evidence, we demonstrate that RNPS1, Y14, SRm160, REF/Aly, TAP, Upf3X and Upf2 are detected in the nuclear fraction on CBP80‐bound but not eIF4E‐bound mRNA. Each of these proteins is also detected on CBP80‐bound mRNA in the cytoplasmic fraction, indicating a presence on mRNA after export. The dynamics of mRNP composition before and after mRNA export are discussed.