title: Intron retention in the alternatively spliced region of RON results from weak 3' splice site recognition.
abstract: The RON gene encodes a tyrosine kinase receptor for macrophage-stimulating protein. A constitutively active isoform that arises by skipping of exon 11 is expressed in carcinomas and contributes to an invasive phenotype. However, a high proportion of the mRNA expressed from the endogenous gene, or from transfected minigenes, appears to retain introns 10 and 11. It is not known whether this represents specific repression or the presence of weak splicing signals. We have used chimeric pre-mRNAs spliced in vitro to investigate the reason for intron retention. A systematic test showed that, surprisingly, the exon sequences known to modulate exon 11 skipping were not limiting, but the 3' splice site regions adjacent to exons 11 and 12 were too weak to support splicing when inserted into a globin intron. UV-crosslinking experiments showed binding of hnRNP F/H just 5' of these regions, but the hnRNP F/H target sequences did not mediate inhibition. Instead, the failure of splicing is linked to weak binding of U2AF65, and spliceosome assembly stalls prior to formation of any of the ATP-dependent complexes. We discuss mechanisms by which U2AF65 binding is facilitated in vivo.
authors.affiliation: Department of Biochemistry, University of Leicester, Leicester, United Kingdom.
authors.forename: Lindsay D
authors.initials: LD
authors.lastname: Smith
authors.forename: Christian M
authors.initials: CM
authors.lastname: Lucas
authors.forename: Ian C
authors.initials: IC
authors.lastname: Eperon
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D001483:Base Sequence; D016384:Consensus Sequence; D003432:Cross-Linking Reagents / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D011485:Protein Binding; D022821:RNA Splice Sites / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D055544:beta-Globins / Q000235:genetics
chemical_list: D003432:Cross-Linking Reagents; D022821:RNA Splice Sites; D012333:RNA, Messenger; D055544:beta-Globins; C080835:RON protein; D020794:Receptor Protein-Tyrosine Kinases