SAM68 binds SMN2 exon 7 and mediates recruitment of hnRNP A1 in vivo. (A) Schematic representation of the human SMN2 gene. Boxes represent exons, black lines represent introns, and the red box indicates the regulated exon 7. Red arrows indicate the oligonucleotide pairs used in the analysis. (B) CLIP assay of SAM68-bound SMN2 pre-mRNA in brain of non-SMA mice (SMN2Δ7;SMN2;Smn^+/+). (C) CLIP assay of hnRNP A1 in brain of non-SMA mice that are wild type or knockout for Sam68. Signals for SAM68 (B) and hnRNP A1 (C) binding was calculated as fold enrichment versus IgGs and expressed as mean ± SD; n = 3. The p-value was determined by two-tailed t test (B) or one-way ANOVA test followed by Bonferroni’s multiple comparison posttest (C). *, P < 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; n.s., not significant (P > 0.05). (D) Sequence of human SMN2 probe used to synthesize the biotinylated RNA for the streptavidin pull-down experiment. Lowercase letters indicate the intron 6 sequence; uppercase letters indicate the exon 7 sequence. Bold letters and red arrows indicate primers used to synthesize the probe, and red bold letters indicate the exonic splicing silencers created by the C to T transition in SMN2 to which SAM68 binds. (E and F) Western blot analysis of the binding of endogenous U2AF65 to the biotinylated probe (SMN2 Ex7) in streptavidin pull-down assays using brain extracts from non-SMA Sam68^+/+ (wt) or Sam68^−/− (ko) mice (E) or extracts from HEK293T cells transfected (+) or not (−) with FLAG-SAM68 (F). Results are representative of three experiments that yielded similar results. ko, knockout; N.E., nuclear extracts; wt, wild type.