FIG. 5.
The zinc finger domains in SF3a60 and SF3a66 are required for their integration into the active 17S U2 snRNP. (A) SF3a purified from HeLa cells (+), buffer (−), or recombinant SF3a subunits, as indicated above the figure, were incubated under splicing conditions with partially purified 15S U2 snRNP in the presence of a 5′-end-labeled oligoribonucleotide complementary to the 5′ end of U2 snRNA. RNP complexes were separated in a native 4% polyacrylamide gel at room temperature and exposed to X-ray film. The positions of the 15S and 17S U2 snRNPs are indicated to the left. (B) Recombinant SF3a subunits were incubated with the 15S U2 snRNP in the combinations shown above the figure and subjected to immunoprecipitation with anti-SF3b155 antibodies coupled to protein G-Sepharose (lanes 6 to 14). Precipitation of the 17S U2 snRNP served as a control (lane 15). Input (lanes 1 to 5) and bound proteins were separated by SDS-PAGE and stained with silver. The positions of SF3b155, SF3b145, and SF3b130 are indicated by a bracket labeled 3b. Recombinant SF3a proteins and SF3a subunits associated with the 17S U2 snRNP are indicated by arrows and lines, respectively. The migration of molecular mass standards (in kilodaltons) is indicated to the left. (C) Derivatives of SF3a60 were incubated with the 15S U2 snRNP and recombinant full-length SF3a66 and SF3a120 as indicated above the figure. The analysis of RNP complexes was performed as in panel A, except that the complexes were resolved at 4°C. (D) Derivatives of SF3a66 were incubated with the 15S U2 snRNP and recombinant full-length SF3a60 and SF3a120 as indicated above the figure. The analysis of RNP complexes was performed as in panel A.