Control of alternative splicing by the differential binding of U1 small nuclear ribonucleoprotein particle

Cellular factors controlling alternative splicing of precursor messenger RNA are largely unknown, even though this process plays a central role in specifying the diversity of proteins in the eukaryotic cell. For the identification of such factors, a segment of the rat preprotachykinin gene was used in which differential expression of neuropeptides gamma and K is dependent on alternative splicing of the fourth exon (E4). Sequence variants of the three-exon segment, (E3-E4-E5) were created, resulting in a sensitive assay for factors mediating the splicing switch between E4-skipping and E4-inclusion. A dinucleotide mutation in the 5' splice site of E4 that increase base-pairing of this site to U1 small nuclear RNA resulted in uniform selection of E4, whereas a control mutation that destroyed base-pairing resulted in uniform E4-skipping. Affinity selection of spliceosomes formed on these functionally distinct substrates revealed that the extreme difference in splicing was mediated by differential binding of the U1 small nuclear ribonucleoprotein particle (snRNP) to the 5' splice site of E4. These data show that, apart from its established role in selecting 5' splice sites, U1 snRNP plays a fundamental role in 3' exon selection and provides insight into possible mechanisms of alternative splicing.