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 w ere transcribed in vitro with 5-fluoro-UTP either partially supplement ing or completely replacing UTP during synthesis. The resulting U2 RNA s have levels of 5-fluorouridine that range from 0 to 100% of the urid ine 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 replacem ent of uridine with 5-FU. However, when the in vitro assembled U2 snRN Ps are subjected to buoyant density gradient centrifugation, the parti cles that contain 100% 5-FU are not resistant to salt dissociation. Wh en the in vitro assembled U2 snRNPs were analyzed by velocity sediment ation gradient centrifugation, 5-FU incorporation correlated with a sh ift in the sedimentation rate of the particles. With 100% 5-FU incorpo ration, the peak of radioactivity shifted to approximately 15 S (contr ol U2 RNA was at approximately 12 S). This peak from 5-FU U2 snRNPs wa s not resistant to dissociation on cesium sulfate gradients. The amoun t of pseudouridine (PSI) found in the RNA from snRNP assembled in vitr o on control and 5-FU-containing U2 RNAs was determined, and even at v ery low levels of 5-FU incorporation (5% replacement), the formation o f PSI was severely inhibited (36% of control). At higher levels of 5-F U incorporation, there was essentially no PSI formed.