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. 2011 Mar 1;25(5):440–444. doi: 10.1101/gad.2009411

Figure 3.

Figure 3.

E7 results in weak binding of U2 snRNP to pre-mRNA. (A) Schematic of the 5′ end of U2 snRNA. The line indicates the 20-nucleotide (nt) oligonucleotide complementary to U2 snRNA used for RNase H cleavage. See the Materials and Methods for sequences of the U2 oligonucleotide and the cntl. (B) Analysis of RNase H-cleaved U2 snRNA. Total RNA from U2 snRNA or cntl extracts was fractionated on a 5% denaturing polyacrylamide gel, and RNA was detected by ethidium bromide. The snRNAs and cleaved U2 snRNA are indicated. (C,D) CMV-Ftz DNA template was incubated in coupled RNAP II transcription/splicing reaction mixtures containing either PlaF (lanes 1,2) or E7 (lanes 3,4) for 5 min in U2 snRNA (ΔU2) or cntl extracts. An aliquot of each sample was mixed with 1.5 μL (Fig. 2C) or 0.5 μL (Fig. 2D) of heparin and fractionated on a 1.2% native agarose gel. (E) Chase assay showing that nonfunctional U2 snRNP remains loosely bound to pre-mRNA after E7 treatment. CMV-Ftz DNA template was transcribed for 5 min in the coupled transcription/splicing system containing 1 μM E7 (lanes 1,2) or PlaF (lanes 3,4). An aliquot (2 μL) of each sample in lanes 1 and 2 was diluted into a new 25-μL reaction mixture in the presence of α-amanitin and incubated for an additional 40 min. For cntl samples in lanes 5–8, CMV-Ftz was transcribed for 5 min in the presence of 100 nM E7107 (lanes 5,6) or 100 nM PlaF (lanes 7,8), α-amanitin was added, and incubation was continued for an additional 40 min. Total RNA was fractionated on a 5% denaturing polyacrylamide gel and detected by PhosphorImager. (F) CMV-Ftz DNA template was incubated in coupled transcription/splicing reactions for 5 min in the presence of E7 or PlaF. Immunoprecipitations were carried out in low salt (250 mM) using a monoclonal antibody to the U2 snRNP B″ protein, a rabbit polyclonal to SF3b155, and monoclonal or polyclonal antibodies to nonrelated proteins as negative controls. Total RNA was run on a 5% denaturing polyacrylamide gel, and CMV-Ftz pre-mRNA was detected by PhosphorImager.