The intramolecular stem-loop structure of U6 snRNA can functionally replace the U6atac snRNA stem-loop

The U6 spliceosomal snRNA forms an intramolecular stem-loop structure durin g spliceosome assembly that is required for splicing and is proposed to be at or near the catalytic center of the spliceosome. U6atac snRNA, the analo g of U6 snRNA used in the U12-dependent splicing of the minor class of spli ceosomal introns, contains a similar stem-loop whose structure but not sequ ence is conserved between humans and plants. To determine if the U6 and U6a tac stem-loops are functionally analogous, the stem-loops from human and bu dding yeast U6 snRNAs were substituted for the U6atac snRNA structure and t ested in an in vivo genetic suppression assay. Both chimeric U6/U6atac snRN A constructs were active for splicing in vivo. In contrast, several mutatio ns of the native U6atac stem-loop that either delete putatively unpaired re sidues or disrupt the putative stem regions were inactive for splicing. Com pensatory mutations that are expected to restore base pairing within the st em regions restored splicing activity. However, other mutants that retained base pairing potential were inactive, suggesting that functional groups wi thin the stem regions may contribute to function. These results show that t he U6atac snRNA stem-loop structure is required for in vivo splicing within the U12-dependent spliceosome and that its role is likely to be similar to that of the U6 snRNA intramolecular stem-loop.