Fig. 5.

Splicing inhibition by the GYF domain. In panels A, B, and D, from the top to the bottom, the position of lariat-splice intermediate, unspliced mRNA, spliced mRNA, and 5′-splice intermediate are indicated. A, inhibition of pre-mRNA splicing by WT GYF domain. BSA was used as negative control, and a C-terminal truncation mutant of the essential splicing factor NIPP1 (NIPP1ΔC) was used as positive control (44). Shown is the image of an in vitro splicing reaction containing body-labeled pre-mRNA after 45 min of incubation under splicing conditions separated on a 10% acrylamide, 7 m urea gel. B, addition of PRS binding mutant (GYF-mut) to an identical in vitro splicing reaction does not inhibit splicing. C, in vitro assembly of spliceosomal complexes. Body-labeled pre-mRNA was incubated in HeLa nuclear extract under standard splicing conditions (lanes 2–5) or in the presence of 70 μm WT GYF (lanes 6–8) and GYF mutant (lanes 9–11), respectively. Spliceosomal complexes (which appear in the order H/E, A, B, and C during spliceosome maturation) present at the indicated time points are shown separated on a 1.5% agarose gel. D, comparison of the GYF effect on the U1-independent splicing of AdML-M3 and U1-dependent splicing of β-globin pre-mRNAs. Prot. conc., protein concentration; IN, input.