Ribonucleoprotein particle assembly and modification of U2 small nuclear RNA containing 5-fluorouridine

An in vitro assembly/modification system was used to study the effect of 5-fluorouridine (5-FU) incorporation on the biosynthesis of the U2 small nuclear ribonucleoprotein particle (U2 snRNP). Labeled U2 RNAs were transcribed in vitro with 5-fluoro-UTP either partially supplementing or completely replacing UTP during synthesis. The resulting U2 RNAs have levels of 5-fluorouridine that range from 0 to 100% of the uridine content. When incubated in reactions containing extracts from HeLa cells, these 5-FU U2 RNAs are assembled into RNPs that are recognized by anti-Sm monoclonal antibody even when there is a complete replacement of uridine with 5-FU. However, when the in vitro assembled U2 snRNPs are subjected to buoyant density gradient centrifugation, the particles that contain 100% 5-FU are not resistant to salt dissociation. When the in vitro assembled U2 snRNPs were analyzed by velocity sedimentation gradient centrifugation, 5-FU incorporation correlated with a shift in the sedimentation rate of the particles. With 100% 5-FU incorporation, the peak of radioactivity shifted to approximately 15 S (control U2 RNA was at approximately 12 S). This peak from 5-FU U2 snRNPs was not resistant to dissociation on cesium sulfate gradients. The amount of pseudouridine (psi) found in the RNA from snRNP assembled in vitro on control and 5-FU-containing U2 RNAs was determined, and even at very low levels of 5-FU incorporation (5% replacement), the formation of psi was severely inhibited (36% of control). At higher levels of 5-FU incorporation, there was essentially no psi formed.