PSEUDOURIDINE FORMATION IN U2 SMALL NUCLEAR-RNA

U2 small nuclear RNA contains 13 pseudouridine (PSI) nucleotides, of which 11 are clustered in 5' regions involved in base-pairing interactions with other RNAs in the spliceosome. As a first step toward understanding the PSI formation pathway in U2 RNA, we investigated PSI formation on unmodified human U2 RNA in a HeLa cell extract system. PSI formation was found to occur specifically within only those RNase T1 oligonucleotide fragments of U2 RNA known to contain PSI in vivo. Using 5-fluorouridine (FUrd)-containing U2 RNAs as specific inhibitors of PSI formation in non-FUrd-substituted substrate U2 RNA, we found that wild-type FUrd-containing U2 RNA as well as several FUrd-containing mutant U2 RNAs completely inhibited PSI formation. In contrast, certain other mutant U2 RNAs containing FUrd displayed reduced inhibitory capacity. In these cases PSI modifications occurred in specific RNase T1 fragments of the substrate U2 RNA only if the FUrd-containing competitor RNA was mutated at or near this site. Formation of PSI at one site in U2 RNA appeared to be neither dependent on prior PSI formation at another site or sites nor required for subsequent PSI formation elsewhere in the molecule. This autonomous mode of PSI formation may be driven by multiple PSI synthase enzymes acting independently at different sites in U2 RNA.