The recruitment of U1 snRNP components to the 5′ss is not decreased by repressive SREs. (A) RNAs were immobilized using Agarose beads and analyzed for the presence of U1 snRNP with specific antibodies directed against U1-70K and U1-C. MS2 coat protein was used as a loading control. N2 represents the splicing near neutral control sequence. (B) HeLa cell nuclear extracts were depleted (D) or mock depleted (U) of functional U1 snRNP using short DNA oligonucleotides and RNase H. The extracts were then used in RNA pulldown assays to demonstrate that U1 snRNA/RNA interactions are necessary for U1 snRNP recruitment. MS2 coat protein and hnRNP A1 were used to control for loading, and N2 represents splicing near neutral control sequences. (C) RNAs containing a muted 5′ss were used in the pulldown assay to demonstrate the effect of a functional or nonfunctional splice site on U1 recruitment.
