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. 2011 May;16(5):479–490. doi: 10.1111/j.1365-2443.2011.01502.x

Figure 4.

Figure 4

TACTAAC repeats in Gomafu delay splicing kinetics in vitro. (A, B) In vitro competition experiments using the Gomafu repeat oligonucleotides. A HeLa cell nuclear extract was pre-incubated on ice, with either water or the oligonucleotides (5 pmol/reaction) used in Fig. 2C. After the addition of the IgM pre-mRNA (A) or AdML pre-mRNA (B), the mixture was incubated at 30 °C for the indicated time. The bands for the RNA products are shown schematically at the right. Note that a marked decrease in the spliced product was observed with the IgM pre-mRNA (dashed box) but not with AdML pre-mRNA. (C) Dose-dependent inhibition of IgM pre-mRNA splicing by the Gomafu repeat oligonucleotides. The in vitro splicing reaction was performed in the presence of water or indicated amount of oligonucleotides at 30 °C for 60 min. Gomafu repeat but not control oligonucleotides inhibited the formation of spliced product in a dose-dependent manner (dashed box). Note that lariat intron is stabilized in the presence of higher amount of oligonucleotides, probably due to an inhibition of endogenous nucleases. (D) Analysis of splicing complex formation. Splicing complexes from the same reaction conditions as in ‘A’ were separated on a native 2% agarose gel. Formation of complex B was significantly retarded in the presence of the Gomafu repeat oligonucleotides. (E) Expression of SF1 in HEK293T cells transfected with control or SF1-expressing vector. Total protein from an equivalent number of cells was separated by 8% SDS–PAGE and detected on the Western blot using anti-FLAG, anti-β-actin and anti-SF1 antibody. The numbers below indicate the relative amount of SF1. (F) Neutralization of inhibitory effect of Gomafu repeat oligonucleotides by an excess amount of SF1. HEK293T cell nuclear extract expressing either empty vector or SF1-FLAG protein was pre-incubated on ice with water or the indicated oligonucleotides (5 pmol/reaction). After the addition of the IgM pre-mRNA, the mixture was incubated at 30 °C for 60 min. Exogenous SF1 protein rescues the splicing efficiency of IgM pre-mRNA (dashed box).