The U1 small nuclear ribonucleoprotein/5' splice site interaction affects U2AF65 binding to the downstream 3' splice site.
In the gene of the neural cell adhesion molecule, the 5' splice site of the alternate exon 18 plays an important role in establishing regulated splicing profiles. To understand how the 5' splice site of exon 18 contributes to splicing regulation, we have investigated the interaction of the U2AF65 splicing factor to pre-mRNAs that contained portions of the constitutive exon 17 or the alternate exon 18 fused to exon 19 and separated by a shortened intron. Despite sharing an identical 3' splice site, only the pre-mRNA that contained a portion of exon 17 and its associated 5' splice site displayed efficient U2AF65 cross-linking. Strikingly, a G-->U mutation at position +6 of the intron, converting the 5' splice site of exon 18 into that of exon 17, stimulated U2AF65 crosslinking. The improved cross-linking efficiency of U2AF65 to a pre-mRNA carrying the 5' splice site of exon 17 required the integrity of the 5' end of U1 but not of U2 small nuclear RNA. Our results indicate that neural cell adhesion molecule 5' splice site sequences influence U2AF65 binding through a U1 small nuclear ribonucleoprotein/U2AF interaction that occurs at the commitment stage of spliceosome assembly, before stable binding of the U2 small nuclear ribonucleoprotein. Thus, the 5' splice sites of exons 17 and 18 differentially affect U2AF65 binding to the 3' splice site of exon 19. Factors that modulate U1 small nuclear ribonucleoprotein binding to these 5' splice sites may play a critical role in regulating exon 18 skipping.
Côté
J
J
Département de Microbiologie, Faculté de Médecine, Université de Sherbrooke, Québec, Canada.
Beaudoin
J
J
Tacke
R
R
Chabot
B
B
D001483:Base Sequence; D017931:DNA Primers; D005091:Exons; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D011485:Protein Binding; D012326:RNA Splicing; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics*; D012261:Ribonucleoproteins / Q000378:metabolism*; D000072668:Splicing Factor U2AF
D017931:DNA Primers; D009687:Nuclear Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; C076753:U2AF1 protein, human