Fig. 3

hnRNP A1 iCLIP reads in the SMN2 gene pinpoint well-known hnRNP A1 binding sites. hnRNP A1 iCLIP identifies well-known hnRNP A1 binding sites in SMN2 exon 7. The intronic splicing silencer (ISS) located at the 3′ splice site. The SMN2-specific exon splicing silencer (ESS) in exon 7, the N1 intronic silencer, and the +100 intronic silencer. a hnRNP A1 iCLIP identifies an hnRNP A1 binding site in SMN2 at the hnRNP A1-dependent silencer in intron 7 + 100. The highly identical SMN1 and SMN2 genes differ at this position (framed: SMN1 + 100A, SMN2 + 100G); previous studies showed the +100G is necessary for strong splicing silencer activity. The iCLIP crosslinking site, i.e., the protein binding site, is assumed to be at the beginning of the sequencing reads (gray) when performing iCLIP. The hnRNP A1 binding peak is represented by a blue line. Only two hnRNP A1 reads were identified in SMN1 at this location (not shown). Screenshot from IGV. b hnRNP A1 iCLIP reads in SMN2 at the hnRNP A1-dependent silencer at the exon 7 3′ splice site (ISS) and at the hnRNP A1-dependent silencer generated by the C > T variation (framed) in SMN2 (ESS). No reads were found in SMN1 at this position (not shown). To ensure that the higher number of hnRNP A1 reads in SMN2 than in SMN1 was not due to higher gene expression, we performed SMN1 and SMN2-specific RT-qPCR (data not shown). This proved that the expression of SMN1 and SMN2 was similar in these cells, and thus was likely not the reason for the higher number of hnRNP A1 reads in SMN2