FIG. 6.

U5 hPrp8 is not properly positioned on the wild-type NRS-Ad3′ RNA. A) The wild-type NRS-Ad 3′ and UU/AA NRS-Ad3′ RNAs were labeled with a single radioactive phosphate between nucleotides G915 and U916, which correspond to positions +1 and +2 relative to the splice junction of the 5′ splice site consensus sequence. The RNAs were incubated in a splicing reaction, UV irradiated to cross-link any bound proteins, and digested with RNase A. The reaction was then denatured and analyzed by SDS-polyacrylamide gel electrophoresis. The sizes of the molecular mass markers are shown on the far left in kilodaltons. The UU/AA NRS-Ad3′ RNA demonstrated the presence of a cross-linked protein of approximately 200 kDa (lane 2, arrow) that was absent from the wild-type NRS-Ad 3′ reaction (lane 1). (B) The UU/AA-Ad3′ RNA was cross-linked as described above and either electrophoresed directly (lane 1) or immunoprecipitated (IP) with an antibody (Ab) against hPrp8 (lane 2) or with no antibody (lane 3).