Pre-mRNA splicing alters mRNP composition: evidence for stable associationof proteins at exon-exon junctions

We provide direct evidence that pre-mRNA splicing alters mRNP protein compo sition. Using a novel in vitro cross-linking approach, we detected several proteins that associate with mRNA exon-exon junctions only as a consequence of splicing. Immunoprecipitation experiments suggested that these proteins are part of a tight complex around the junction. Two were identified as SR m160, a nuclear matrix-associated splicing coactivator, and hPrp8p, a core component of U5 snRNP and spliceosomes. Glycerol gradient fractionation sho wed that a subset of these proteins remain associated with mRNA after its r elease from the spliceosome. These results demonstrate that the spliceosome can leave behind signature proteins at exon-exon junctions. Such proteins could influence downstream metabolic events in vivo such as mRNA transport, translation, and nonsense-mediated decay.