Fig. 5

SSOs targeting hnRNP A1 binding sites downstream of the MTRR pseudoexon improve pseudoexon inclusion. a Model of the hnRNP A1-mediated repression of the MTRR pseudoexon based on the hnRNP A1 iCLIP reads. The significant hnRNP A1 binding peak downstream of the 5′ splice site is indicated. hnRNP A1 may bind downstream of the 5′ splice site to repress splice site recognition by U1 snRNP. The target site for the MTRR SSO (green) covers three hnRNP A1 binding motifs. b Western blot with hnRNP A1 or as control TDP43 antibody of proteins purified by RNA-affinity chromatography of biotin-conjugated RNA oligonucleotides covering the downstream region of the MTRR 5′ splice site. Disruption of the hnRNP A1 binding motifs reduces hnRNP A1 binding. The motifs are scored using our generated scoring matrix (Additional file 1: Figure S1). The proximal hnRNP A1 motif is required for hnRNP A1 binding. hnRNP A1 binding motifs are underscored, and mutations disrupting the motifs are red. Representative of three experiments. c RT-qPCR analysis of the inclusion of the endogenous MTRR pseudoexon in control and hnRNP A1 knockdown HeLa cells. MTRR pseudoexon inclusion increases after hnRNP A1 knockdown. d RT-PCR of SSO-treated HeLa or Hek293 cells. SSO-mediated blocking of the hnRNP A1 binding sites near the MTRR pseudoexon improves endogenous MTRR pseudoexon inclusion. SSO transfections were done in duplicate and gel bands were semi-quantified using ImageJ. PSI percent spliced in