SMN2 exon 7 skipping is associated with spinal muscular atrophy and has been a major therapeutic target. However, the mechanism regulating exon 7 splicing remains largely unknown. This study uncovered that hnRNPR potently inhibits exon 7 inclusion through binding to an AU-rich element of the exon. Both hnRNPR and Sam68 bind to the element in a competitive manner and the inhibitory effect of hnRNPR is much stronger than Sam68. Moreover, we found that ASOs inducing hnRNPR exon 5 skipping generate a less inhibitory isoform and promote SMN2 exon 7 inclusion. Therefore, we identified a novel mechanism underlying SMN2 mis-splicing. (By Tao Jiang, https://jmg.bmj.com/content/early/2023/05/24/jmg-2023-109186 )