Metabolically labeled NEs retained full catalytic activity, as shown by analyzing the B and C complexes purified from “heavy” or “light” SILAC NEs, respectively. (A) The splicing kinetics were determined from aliquots of splicing reactions taken from 0–180 min and analyzed by denaturing gel electrophoresis. Pre-mRNA and splicing products were visualized by autoradiography. Splicing products first appeared after 10 min. (B) The spliceosomal complex formation was assayed by native agarose gel electrophoresis and visualized by autoradiography. The A and B complexes were first observed after 2 and 4 min, respectively, while the C complex first appeared after 10–15 min. (C) The RNA compositions of purified B (“light” SILAC NE) and C (“heavy” SILAC NEs) complexes were analyzed by denaturing gel electrophoresis and visualized by silver staining (lanes 1,3) or autoradiography (lanes 2,4). B complexes contained U1, U2, U4, U5, and U6 snRNA (lane 1) and a large amount of pre-mRNA (lane 2). C complexes contained U2, U5, and U6 snRNA (lane 3) and splicing products and reduced amounts of pre-mRNA (lane 4).
