Figure 6.

Contributions of the Quadruplex and hnRNP F/H to the Activity of GGA

(A and B) Triplicate assays of splicing in vitro of SMN2 exon 7-containing RNA were done in the presence or absence of 250 nM GGA. The quadruplex-stabilizing molecules GSA-0820 and GSA-0902 were dissolved in DMSO and added to the reactions to produce the final concentrations shown. The pre-mRNA and inclusion and exclusion mRNA products are indicated. Charts to the right of each phosphorimage show the ratio of inclusion/exclusion of exon 7 at each of the concentrations tested. Error bars show the SDs of the ratios for the triplicates.

(C) Time courses of splicing in vitro in the nuclear extracts used in Figure 4E. The extracts were prepared from HEK293T cells transfected with plasmids expressing mEGFP-hnRNP F and mEGFP-hnRNP H (+F/H) or siRNA targeting hnRNP F and H (F/H RNAi). −, mock transfection. Reactions were done in the presence or absence of GGA and samples taken at 0, 0, 60, and 120 min. The underlined lanes were transposed in the image.

(D) Analysis by western blotting of the nuclear extracts used for the experiments in Figures 4E and 6C. Antibodies detecting hnRNP F and H and U1A were detected by fluorescent secondary antibodies. C, mock transfection; −, untransfected.

See also Figures S2 and S3.