FIGURE 5.

5′SS-Mediated splicing inhibition and commitment to splicing pathway. (A) Transcription does not facilitate recruitment of splicing factors from complexes that form on 5′SS RNA oligonucleotides. Splicing (left panel) or transcription–splicing reactions (right panel) were either carried out normally (lanes 1,2,5,6) or preincubated for 15 min in the presence (lanes 4,8) or absence of 5′SS RNA oligonucleotide (lanes 3,7). Following preincubation either β-globin RNA or CMV-β globin DNA template was added and the samples were incubated further for a total of 2 h. Precursors, products, and intermediates of the splicing reactions are indicated to the left of the panel, and those of the transcription–splicing reactions are indicated to the right of the panel. (B) Commitment of presynthesized RNA to the splicing pathway. Splicing reactions were allowed to proceed for 5, 10, or 15 min; the incubations were interrupted and 5′SS RNA oligonucleotide was added to 5 μM final concentration, and the samples were incubated further for a total of 2 h. 5′SS RNA was added to one of the reactions at the start of the incubation (0′). Precursors, products, and intermediates of the splicing reactions are indicated to the left of the panel. (C). Same assay as in B with transcription–splicing reactions. Reactions were carried out for the indicated periods of time and were either stopped or the incubation was interrupted and α-amanitin was added to a final concentration of 1 ng/μL or α-amanitin and 5′SS RNA oligonucleotide were added together to 1 ng/μL and 5 μM final concentrations, respectively. Samples were incubated further for a total of 2 h. 5′SS RNA was added to one of the reactions at the start of the incubation (0′). Precursors, products, and intermediates of the transcription splicing reactions are indicated to the left of the panel.