Effect of 5-FU substitution and mutation on Sm protein binding to human U4 snRNA

The effects of native and non-native nucleotide substitution on the binding of Sm proteins to human U4 snRNA were investigated to determine if the Sm site was a likely target for the RNA-mediated effects of the anticancer drug 5-FU, and other nucleoside analogues. The Sm binding site of human U4 snRNA was prepared by in vitro transcription, and Sm protein binding was assessed using gel mobility shift assays. The U4:Sm RNA:protein complex was identified by immunoprecipitation with the Sm-specific Y12 antibody. The effects of 5-FU substitution were assessed by including FUTP in the in vitro transcription reactions. The effects of native nucleotide substitution were assessed by mutagenesis. Deletion mutants were used to assess the relative importance of the two stem-loops that flank the Sm binding site for protein binding. Point mutation (U-->G) to the 5'-Urd in the Sm site reduced Sm protein binding while similar point mutation to the 3'-Urd had a lesser effect. Mutation (U-->G) of all Urd in the Sm site completely inhibited Sm protein binding. The central stem-loop contributed significantly to Sm protein complex formation but the 3' stem-loop had little effect. Substitution of Urd by 5-fluorourdine (FUrd) did not inhibit Sm protein binding, but reduced the stability of the resulting complex. The results indicate that 5-FU, or other Uracil analogues, are unlikely to exert RNA-mediated effects through inhibition of Sm protein binding.