In the course of analyzing 5′ splice site mutations in the second intron of Schizosaccharomyces pombe cdc2, we identified a cryptic 5′ junction containing a nonconsensus nucleotide at position +2. An even more unusual feature of this cryptic 5′ junction was its pattern of activation. By analyzing the profile of splicing products for an extensive series of cdc2 mutants in the presence and absence of compensatory U1 alleles, we have obtained evidence that the natural 5′ splice site participates in activation of the cryptic 5′ splice site, and that it does so via base pairing to U1 snRNA. Furthermore, the results of follow-up experiments strongly suggest that base pairing between U1 snRNA and the cryptic 5′ junction itself plays a dominant role in its activation. Most remarkably, a mutant U1 can activate the cryptic 5′ splice site even in the presence of a wild-type sequence at the natural 5′ junction, providing unambiguous evidence that this snRNA redirects splicing via base pairing. Although previous work has demonstrated that U5 and U6 snRNAs can activate cryptic 5′ splice sites through base pairing interactions, this is the first example in which U1 snRNA has been implicated in the final selection of a cryptic 5′ junction.

The rat β-tropomyosin (β-TM) gene encodes both skeletal muscle β-TM mRNA and nonmuscle TM-1 mRNA via alternative RNA splicing. This gene contains eleven exons: exons 1–5, 8, and 9 are common to both mRNAs; exons 6 and 11 are used in fibroblasts as well as in smooth muscle, whereas exons 7 and 10 are used in skeletal muscle. Previously we demonstrated that utilization of the 3′ splice site of exon 7 is blocked in nonmuscle cells. In this study, we use both in vitro and in vivo methods to investigate the regulation of the 5′ splice site of exon 7 in nonmuscle cells. The 5′ splice site of exon 7 is used efficiently in the absence of flanking sequences, but its utilization is suppressed almost completely when the upstream exon 6 and intron 6 are present. The suppression of the 5′ splice site of exon 7 does not result from the sequences at the 3′ end of intron 6 that block the use of the 3′ splice site of exon 7. However, mutating two conserved nucleotides GU at the 5′ splice site of exon 6 results in the efficient use of the 5′ splice site of exon 7. In addition, a mutation that changes the 5′ splice site of exon 7 to the consensus U1 snRNA binding site strongly stimulates the splicing of exon 7 to the downstream common exon 8. Collectively, these studies demonstrate that 5′ splice site competition is responsible, in part, for the suppression of exon 7 usage in nonmuscle cells.

Selection of pre-mRNA splice sites is a highly accurate process involving many trans-acting factors. Recently, we described a role for U6 snRNA position G52 in selection of the first intron nucleotide (+1G). Because some U2 alleles suppress U6-G52 mutations, we investigated whether the corresponding U2 snRNA region also influenced 5′ splice site selection. Our results demonstrate that U2 snRNAs mutated at position U23, but not adjacent nucleotides, specifically affect 5′ splice site cleavage. Furthermore, all U2 position U23 mutations are synthetic lethal with the thermosensitive U6-G52U allele. Interestingly, the U2-U23C substitution has an unprecedented hyperaccurate splicing phenotype in which cleavage of introns with a +1G substitution is reduced, whereas the strain grows with wild-type kinetics. U2 position U23 forms the first base pair with U6 position A59 in U2/U6 helix Ib. Restoration of the helical structure suppresses 5′ splice site cleavage defects, showing an important role for the helix Ib structure in 5′ splice site selection. U2/U6 helix Ib and helix II have recently been described as being functionally redundant. This report demonstrates a unique role for helix Ib in 5′ splice site selection that is not shared with helix II.

Nuclear pre-mRNA splicing necessitates specific recognition of the pre-mRNA splice sites. It is known that 5′ splice site selection requires base pairing of U6 snRNA with intron positions 4–6. However, no factor recognizing the highly conserved 5′ splice site GU has yet been identified. We have tested if the known U6 snRNA–pre-mRNA interaction could be extended to include the first intron nucleotides and the conserved 50GAG52 sequence of U6 snRNA. We observe that some combinations of 5′ splice site and U6 snRNA mutations produce a specific synthetic block to the first splicing step. In addition, the U6-G52U allele can switch between two competing 5′ splice sites harboring different nucleotides following the cleavage site. These results indicate that U6 snRNA position 52 interacts with the first nucleotide of the intron before 5′ splice site cleavage. Some combinations of U6 snRNA and pre-mRNA mutations also blocked the second splicing step, suggesting a role for the corresponding nucleotides in a proofreading step before exon ligation. From studies in diverse organisms, various functions have been ascribed to the conserved U6 snRNA 47ACAGAG52 sequence. Our results suggest that these discrepancies might reflect variations between different experimental systems and point to an important conserved role of this sequence in the splicing reaction.