Figure 2.

RNAP II transcription is functionally coupled to spliceosome assembly. (A) Nuclear extract was incubated under transcription/splicing conditions for the indicated times in the absence of DNA template (lanes 1,2) or presence of CMV-DoA1 Δ5′Δ3′ DNA template (lanes 3,4) or CMV-DoA1 DNA template (lanes 5,6). Heparin was added to each sample, which was then loaded on a 1.2% low-melting-point agarose mini-gel. Bands were detected by PhosphorImager. (B) RNA was extracted from the indicated regions (i–v) of the gel shown in A and fractionated on an 8% denaturing polyacrylamide gel (lanes 2,4–7); 15 and 60 min transcription/splicing reactions were run as markers (lanes 1,3). (C) Naked DoA T7 pre-mRNA or the corresponding CMV-DoA DNA template was incubated under transcription/splicing conditions for the times indicated. For the CMV-DoA DNA template, Actinomycin D was added at time 0 and incubation continued for 60 min (lane 5), or Actinomycin D was added at 15 min and incubation was continued for 0 min (lane 6), 15 min (lane 7), or 45 min (lane 8). Heparin was added to each sample, which were then fractionated side-by-side on a 1.2% low-melting-point agarose gel. The spliceosome, spliced mRNP, and nonspecific H complex are indicated. (D) DoA CMV DNA template was incubated under transcription/splicing conditions for the indicated times, and total RNA was fractionated on an 8% polyacrylamide gel. (E) DoA CMV DNA template was incubated under transcription/splicing conditions for the indicated times; heparin was added to each sample, which was then run on a 1.2% low-melting-point agarose gel. (F) T7 DoF DNA template with T7 polymerase or corresponding CMV-DoF DNA template was incubated under transcription/splicing conditions for the indicated times; heparin was added to each sample, which was then fractionated on a 1.2% low-melting point agarose gel. (G) RNA was extracted from the bands indicated (left of i–iii) of the gel shown in F and fractionated on an 8% denaturing polyacrylamide gel.