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. 2013 Jul;19(7):971–981. doi: 10.1261/rna.038638.113

FIGURE 1.

FIGURE 1.

The spliceosome catalyzes the debranching reaction. (A) Splicing reaction was carried out in Prp16-depleted Cwc25-HA extracts, and the spliceosome was precipitated with anti-HA antibody. The purified spliceosome was incubated in 10 mM Tris-HCl, at pH 6.8, 7.4, 8.0, or 8.8, 4 mM MgCl2 without (lanes 3–6) or with 150 mM KCl (lanes 7–10) or 150 mM NaCl (lanes 11–14). RNA isolated as in lane 2 was also incubated in 10 mM Tris-HCl, pH 8.8, 4 mM MgCl2, and 150 mM of KCl without (lane 15) or with100 nM recombinant Dbr1 (lane 16). (B) The lariat (lane 1) and putative linear (lane 2) intron-exon 2 RNA were gel-purified and analyzed by primer extension with oligonucleotides A7. (C) The spliceosome precipitated with anti-Ntc20 antibody was incubated in 10 mM Tris-HCl, pH 8.8, 4 mM MgCl2 in the absence (lanes 3,4) or presence of 150 mM NaCl (lanes 5,6) for 1 h. The spliceosome was then washed with 1 mL 10 mM Tris-HCl, pH 8.8, and reincubated in 10 mM Tris-HCl, pH 8.8, 4 mM MgCl2 in the absence (lanes 3,5) or presence of 150 mM KCl (lanes 4,6) for 1 h. (D) The spliceosome formed in dbr1Δ extracts was incubated in 10 mM Tris-HCl, pH 8.8, 4 mM MgCl2, and 150 mM KCl for 1 h (lane 3), and the supernatant (lane 5) was separated from beads (lane 4). (E) A splicing reaction was carried out in Prp16-depleted extracts using wild-type (lanes 1–5) or ACAC (lanes 6–10) pre-mRNAs with high (lanes 1–3, 6–8) or low radioactivity (lanes 4,5,9,10), and the spliceosomes were precipitated with anti-Ntc20 antibody. Spliceosomes assembled on wild-type pre-mRNA (lane 2) were incubated in 10 mM Tris-HCl, pH 8.8, 4 mM MgCl2, and 150 mM KCl for 1 h (lanes 3–5), and RNA was isolated. RNA was also isolated from spliceosomes assembled on ACAC pre-mRNA and treated with Dbr1 (lanes 8–10). Purified RNA was subjected to phosphorylation using polynucleotide kinase and γ-32P-ATP without (lanes 4,9) or with (lanes 5,10) pretreatment with calf intestine alkaline phosphatase. (R) 1/10 of reaction mixture, (SP) spliceosome, (DBR) debranching reaction catalyzed by the spliceosome, (Dbr1) debranching reaction catalyzed by the debranchase.