In metazoans, splicing of introns from pre-mRNAs can occur by two pathways: the major U2-dependent or the minor U12-dependent pathways. Whereas the U2-dependent pathway has been well characterized, much about the U12-dependent pathway remains to be discovered. Most of the information regarding U12-type introns has come from in vitro studies of a very few known introns of this class. To expand our understanding of U12-type splicing, especially to test the hypothesis that the simple base-pairing mechanism between the intron and U12 snRNA defines the branchpoint of U12-dependent introns, additional in vitro splicing substrates were created from three putative U12-type introns: the third intron of the Xenopus RPL1a gene (XRP), the sixth intron of the Xenopus TFIIS.oA gene (XTF), and the first intron of the human Sm E gene (SME). In vitro splicing in HeLa nuclear extract confirmed U12-dependent splicing of each of these introns. Surprisingly, branchpoint mapping of the XRP splicing intermediate shows use of the upstream rather than the downstream of two consecutive adenosines within the branchpoint sequence (BPS), contrary to the prediction based on alignment with the sixth intron of human P120, a U12-dependent intron whose branch site was previously determined. Also, in the SME intron, the position of the branchpoint A residue within the region base paired with U12 differs from that in P120 and XTF. Analysis of these three additional introns therefore rules out simple models for branchpoint selection by the U12-type spliceosome.

Two forms of spliceosomes were found in higher eukaryotes. The major form contains the U1, U2, U4, U5, and U6 snRNAs; the minor form contains the U11, U12, U4atac, U5, and U6atac snRNAs. Assembly and function of the major form are based on a complex dynamic of UsnRNA–UsnRNA and UsnRNA–pre-mRNA interactions, and the involved UsnRNA segments are highly posttranscriptionally modified in plants and vertebrates. To further characterize the minor form of spliceosomes, we looked for the Ψ residues in HeLa cells' U11, U12, U4atac, and U6atac snRNAs, using chemical approaches. Four Ψ residues were detected in total for these four atac UsnRNAs, compared to 20 in their counterparts of the major spliceosomes. The two Ψ residues detected in U12 are also found in U2 snRNA. One of them belongs to the branch-site-recognition sequence. It forms one of the base pairs that bulge out the A residue, responsible for the nucleophilic attack. Conservation of this strategic Ψ residue probably reflects a functional role. Another Ψ residue was detected in a U4atac snRNA segment involved in formation of helix II with U6atac. The fourth one was detected in the additional stem-loop structure present at the 3′ end of U6atac snRNA. Differences in Ψ content of the atac and major UsnRNAs of human cells may participate in the differentiation of the two splicing systems. Based on secondary structure similarity, U2 and U12 snRNAs on the one hand and U4 and U4atac snRNAs on the other hand may share common Ψ synthases.