Pseudouridines in and near the branch site recognition region of U2 snRNA are required for snRNP biogenesis and pre-mRNA splicing in Xenopus oocytes

Virtually all uridines in the branch site recognition region (BSRR) of vertebrate U2 are converted into pseudouridines after initial transcription. Here, we report a functional analysis of these modified nucleotides using the Xenopus oocyte reconstitution system. Using site-specific P-32-labeling and TLC, we show that U2 pseudouridylation occurs much faster in the BSRR than in the 5'-terminal region. To functionally dissect the pseudouridines in the BSRR, we replaced each uridine with 5-fluorouridine (unmodifiable nucleotide) using site-specific RNase H cleavage directed by 2'-O-amethyl-RNA-DNA chimeras followed by three-piece ligation. Whereas in vitro transcribed U2 containing no 5-fluorouridines rescued splicing in U2-depleted oocytes, no rescue was observed with U2 RNA containing 5-fluorouridines introduced into the BSRR. Additionally, U2 RNA containing 5-fluorouridines in the BSRR specifically inhibited pseudouridylation in the BSRR of in vitro transcribed U2 injected at a later time, although pseudouridylation in the 5'-end region was not affected. Our reconstitution results indicated that prior injection into U2-depleted oocytes with U2 RNA containing 5-fluorouridines in the BSRR almost completely abrogated the ability of in vitro transcribed U2 to rescue splicing, whereas full rescue was obtained with either cellular U2 or U2 containing pseudouridines in the BSRR. Further analyses using glycerol-gradient and native gel electrophoresis indicated that U2 RNAs lacking the BSRR pseudouridines do not participate in the assembly of the functionally active 17S U2 snRNP and the spliceosome. We conclude that the BSRR pseudouridines of vertebrate U2 are required for complete snRNP assembly and pre-mRNA splicing in Xenopus oocytes.