Base pairing between the 5′ end of U1 snRNA and the conserved 5′ splice site of pre-mRNA is important for commitment complex formation in vitro. However, the biochemical mechanisms by which pre-mRNA is initially recognized by the splicing machinery is not well understood. To evaluate the role of this base pairing interaction, we truncated U1 snRNA to eliminate the RNA–RNA interaction and surprisingly found that U1 snRNP can still form a nearly normal RNA–protein complex and maintain sequence specificity. We propose that some feature of U1 snRNP, perhaps one or more protein factors, is more important than the base pairing for initial 5′ splice site recognition. In addition, at least five sets of interactions contribute to complex formation or stability. Only one of these is base pairing between the 5′ splice site and the 5′ end of U1 snRNA, without which the U1 snRNP–pre-mRNA complex is less stable and has a somewhat altered conformation.

The minor U12-dependent class of eukaryotic nuclear pre-mRNA introns is spliced by a distinct spliceosomal mechanism that requires the function of U11, U12, U5, U4atac, and U6atac snRNAs. Previous work has shown that U11 snRNA plays a role similar to U1 snRNA in the major class spliceosome by base pairing to the conserved 5′ splice site sequence. Here we show that U6atac snRNA also base pairs to the 5′ splice site in a manner analogous to that of U6 snRNA in the major class spliceosome. We show that splicing defective mutants of the 5′ splice site can be activated for splicing in vivo by the coexpression of compensatory U6atac snRNA mutants. In some cases, maximal restoration of splicing required the coexpression of compensatory U11 snRNA mutants. The allelic specificity of mutant phenotype suppression is consistent with Watson–Crick base pairing between the pre-mRNA and the snRNAs. These results provide support for a model of the RNA–RNA interactions at the core of the U12-dependent spliceosome that is strikingly similar to that of the major class U2-dependent spliceosome.