Natural base-pairing interactions between 5' splice site and branch site sequences affect mammalian 5' splice site selection.
In the murine gene encoding the neuronal cell adhesion molecule (NCAM), the integrity of the 5' splice site of exon 18 (E18) is essential for regulation of alternative splicing. To further study the contribution of 5' splice site sequences, we used a simple NCAM pre-mRNA containing a portion of E18 fused to E19 and separated by a shortened intron. This RNA is spliced in vitro to produce five sets of lariat intermediates and products, the most abundant set displaying aberrant migration in acrylamide/urea gels. Base pairing interactions between positions +5 and +8 of the intron and positions -3 and -6 from the branch point were responsible for the faster migration of this set of lariat molecules. To test whether the duplex structure forms earlier and contributes to 5' splice site selection, we used NCAM substrates carrying the 5' splice sites of E17 and E18 in competition for the 3' splice site of E19. Mutations upstream of the major branch site improve E18/E19 splicing in NIH3T3 extracts, whereas compensatory mutations at positions +7 and +8 neutralize the effect of branch site mutations and curtail E18/E19 splicing. Our data suggest that duplex formation occurs early and interferes with the assembly of complexes initiated on the 5' splice site of NCAM E18. This novel type of intron interaction may exist in the introns of other mammalian pre-mRNAs.
Côté
J
J
Département de Microbiologie et d'Infectiologie, Faculté de Médecine, Université de Sherbrooke, Québec, Canada.
Chabot
B
B
D016475:3T3 Cells; D017398:Alternative Splicing; D000818:Animals; D001482:Base Composition; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007438:Introns; D051379:Mice; D016297:Mutagenesis, Site-Directed; D019006:Neural Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins; D011485:Protein Binding; D012322:RNA Precursors / Q000737:chemistry / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D000072668:Splicing Factor U2AF
D019006:Neural Cell Adhesion Molecules; D009687:Nuclear Proteins; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C000607494:U2AF2 protein, human; C000607495:Zrsr1 protein, mouse