Abstract
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.
Highlights
The first step of splicing of nuclear pre-mRNAs occurs through cleavage at the 5' splice site with concomitant joining of the intron 5' end to the 2' hydroxyl of a residue in the branch site region of the intron
To gain a better understanding of the molecular mechanisms involved in exon 18 skipping, we have investigated RNA-protein interactions on pre-mRNA substrates carrying various 5' splice sites
As th e 5' end of U1 snRNA is ess ent ia l for U1 s nRNP binding to 5' splice site seque nces (3 1), our results indicate tha t a U1 s nRNP/5' splice site interaction is required for' efficie nt U2AF B5 cross-linking to D RNA
Summary
The branch site region is recognized by the V2 snRNP [5] and requires prior binding of the non-snRNP splicing factor V2AF65 to the downstream polypyrimidine tract-AG at the 3' splice junction [6, 7]. 5' splice site sequences downstream from a weak 3' splice site have been shown to facilitate V2AF65 binding in a process that requires the participation of other factoris) [17]. A similar network of interactions involving VI snRNP and V2AF is possibly assembled across the exon to stimulate the use of a weak 3' splice site. To gain a better understanding of the molecular mechanisms involved in exon 18 skipping, we have investigated RNA-protein interactions on pre-mRNA substrates carrying various 5' splice sites. 5' splice site of exon 18 into that of exon 17 improves the interaction of U2AF65 with the 3' splice site, in a process that requires the U1 but not the U2 snRNP
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