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The U1 small nuclear ribonucleoprotein/5' splice site interaction affects U2AF65 binding to the downstream 3' splice
1Département de Microbiologie, Faculté de Médecine, Université de Sherbrooke, Québec, Canada.
The Journal of Biological Chemistry
|February 24, 1995
Summary
The 5' splice site of neural cell adhesion molecule exon 18 influences splicing regulation by affecting U2AF65 binding. This interaction, mediated by U1 small nuclear ribonucleoprotein, occurs early in spliceosome assembly, impacting exon 18 skipping.
Area of Science:
- Molecular Biology
- RNA Splicing
- Gene Regulation
Background:
- The neural cell adhesion molecule (NCAM) gene features an alternate exon 18 with a critical 5' splice site influencing splicing patterns.
- Understanding the mechanisms governing alternative splicing is crucial for comprehending gene expression regulation.
Purpose of the Study:
- To investigate how the 5' splice site of NCAM exon 18 contributes to regulated splicing.
- To elucidate the interaction between the U2AF65 splicing factor and pre-mRNAs containing sequences from exons 17 or 18.
Main Methods:
- Investigated U2AF65 splicing factor interaction with pre-mRNAs containing exon 17 or 18 fused to exon 19.
- Utilized cross-linking assays to assess U2AF65 binding efficiency.
- Performed site-directed mutagenesis to alter the 5' splice site sequence.
Main Results:
- Efficient U2AF65 cross-linking was observed only with pre-mRNA containing the exon 17 5' splice site, not exon 18.
- A G to U mutation at intron position +6, mimicking the exon 17 splice site, significantly enhanced U2AF65 cross-linking to exon 18 pre-mRNA.
- Enhanced U2AF65 binding required intact U1 small nuclear RNA but not U2 small nuclear RNA.
Conclusions:
- NCAM 5' splice site sequences modulate U2AF65 binding through a U1 small nuclear ribonucleoprotein/U2AF interaction during early spliceosome assembly.
- This interaction occurs before stable U2 small nuclear ribonucleoprotein binding, influencing the commitment stage.
- Differential effects of exon 17 and 18 5' splice sites on U2AF65 binding to exon 19's 3' splice site suggest a role for U1 binding factors in regulating exon 18 skipping.