hnRNP A1 and hnRNP F Modulate the Alternative Splicing of Exon 11 of the Insulin Receptor Gene
Figure 2
hnRNP A1 and -F/H and SRSF1 bind to G-rich motifs in intron 10.
Panel A: RNA-affinity purification of HeLa nuclear proteins using templates IE3 or IE3m, which contains the GA7 mutation. Sequences of IE3 and IE3m are given at top. Bound proteins were washed four times with 100 mM KCl and then eluted with increasing concentrations of KCl. Proteins from washes 1 and 4 and from the KCl elution were separated on a 4–12% Bis-Tris gel and stained with Coomassie blue. Arrowheads indicate differential protein bands between the two RNA templates. Panel B: In vitro RNA pull-down assay using RNA templates IE1, IE2 and IE3 and their cognate mutants. Bound proteins were separated by gel electrophoresis and immunoblotted for hnRNP A1, hnRNP H, hnRNP F or SRSF1. Panel C: Summary of binding to 5′ GA-rich motifs. The consensus binding sites for hnRNP F [34], hnRNP A1 [35], and SRSF1 [36] are shown. Relative binding associated with the RNA is indicated by plus signs. Minus signs denote no detectable binding. Panel D: Summary of binding to 3′ GA-rich motifs. The consensus binding sites for hnRNP F and hnRNP A1 are shown. Relative binding associated with the RNA is indicated by plus signs. Minus signs denote no detectable binding.