A NOVEL U2-U6 SNRNA STRUCTURE IS NECESSARY FOR MAMMALIAN MESSENGER-RNA SPLICING

Splicing of mRNA precursors requires a complex and dynamic set of RNA- RNA base-pairing interactions in which the U2 and U6 snRNAs play centr al roles. Using a genetic suppression assay, we refine and extend a U2 -U6 snRNA structure that may comprise the catalytic center of the spli ceosome. We first show that a critical U2-U6 helix proven in yeast, he lix Ia, is also essential for mammalian splicing. Mutations in the adj acent helix Ib, however, cannot be similarly suppressed, and relevant residues in both U2 and U6 are shown to participate in intramolecular, rather than intermolecular, base-pairing. We next demonstrate the req uirement for a novel U2-U6 helix, helix III, which involves bases exte nding 3' from the branch site recognition sequence in U2 and 5' from a n evolutionarily invariant sequence in U6 implicated previously in 5' splice site recognition. This configuration suggests that helix III ma y help juxtapose the pre-mRNA 5' splice site and branch site. We provi de evidence for this by demonstrating that a branch site mutation can be suppressed by a mutation in the 5' splice site, provided that compe nsatory changes are made in the appropriate bases in U2 and U6. Our re sults provide new insights into how U2 and U6 snRNAs interact with eac h other and with the pre-mRNA to initiate the first catalytic step in splicing.