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Fig. 4 | BMC Biology

Fig. 4

From: Global identification of hnRNP A1 binding sites for SSO-based splicing modulation

Fig. 4

SSOs targeting an hnRNP A1 binding site downstream of SKA2 exon 3 improve exon inclusion. a RT-PCR of SKA2 exon 3 splicing in HeLa cells with (KD) or without (Ctrl) hnRNP A1 knockdown. hnRNP A1 knockdown increases SKA2 exon 3 inclusion. PSI percent spliced in; semi-quantification of bands on agarose gels by ImageJ. b Western blot of proteins purified by RNA-affinity chromatography of biotin-conjugated RNA oligonucleotides covering the three putative hnRNP A1 binding sites near the 5′ splice site of SKA2 exon 3. hnRNP A1 binding motifs are underscored, and mutations disrupting the hnRNP A1 binding motifs are shown in red. Introduction of the mutations reduces hnRNP A1 binding, while modifying one of the hnRNP A1 motifs to match our generated consensus binding motif (green) improves hnRNP A1 binding. The score of the putative hnRNP A1 motifs were calculated based on our generated scoring matrix (Additional file 1: Figure S1). Representative of two experiments. c hnRNP A1 iCLIP reads at the 5′ splice site of SKA2 exon 3. hnRNP A1 may bind downstream of the 5′ splice site to repress 5′ splice site recognition. The target site for the SKA2 exon 3 SSO is shown in green. The gene is on the antisense strand. Screenshot from IGV. d Transfection of SSOs targeting the hnRNP A1 binding site downstream of the 5′ splice site in SKA2 exon 3 into HeLa, HEK293, or A549 cells improves exon inclusion. The intensity of the bands was semi-quantified using ImageJ. PSI percent spliced in

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