Figure 5.

MAH is present in the in vitro-assembled pre-mRNA splicing complex. HeLa nuclear extract was incubated with ³²P-labeled PIP85A splicing substrate for 40 min at 30°C, and then equal volumes of splicing complexes were immunoprecipitated with the indicated antibodies. RNAs were separated on 15% polyacrylamide/urea gel. Lanes 1, in vitro-transcribed PIP85A pre-mRNA; 2, molecular weight marker, [γ-³²P]ATP end-labeled 100 bp DNA ladder; 3, total splicing reaction without immunoprecipitation, half of assay reaction than in lanes 4–7. Ctr Ab, preimmune serum (lane 4); C12, anti-MAH antiserum (lane 5); Sm, anti-Sm antibody (lane 6); B1C8, anti-B1C8 antibody (lane 7). Ctr and C12 are equal to 150 μg IgG. , pre-mRNA; , lariat intermediate; , lariat product; , spliced mRNA; , intron.