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Original research
HnRNPR strongly represses splicing of a critical exon associated with spinal muscular atrophy through binding to an exonic AU-rich element


Background Spinal muscular atrophy (SMA) is a motor neuron disease caused by mutations of survival of motor neuron 1 (SMN1) gene, which encodes the SMN protein. SMN2, a nearly identical copy of SMN1, with several single-nucleotide substitutions leading to predominant skipping of its exon 7, is insufficient to compensate for loss of SMN1. Heterogeneous nuclear ribonucleoprotein R (hnRNPR) has been previously shown to interact with SMN in the 7SK complex in motoneuron axons and is implicated in the pathogenesis of SMA. Here, we show that hnRNPR also interacts with SMN1/2 pre-mRNAs and potently inhibits exon 7 inclusion.

Methods In this study, to examine the mechanism that hnRNPR regulates SMN1/2 splicing, deletion analysis in an SMN2 minigene system, RNA-affinity chromatography, co-overexpression analysis and tethering assay were performed. We screened antisense oligonucleotides (ASOs) in a minigene system and identified a few that markedly promoted SMN2 exon 7 splicing.

Results We pinpointed an AU-rich element located towards the 3′ end of the exon that mediates splicing repression by hnRNPR. We uncovered that 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, among the four hnRNPR splicing isoforms, the exon 5-skipped one has the minimal inhibitory effect, and ASOs inducing hnRNPR exon 5 skipping also promote SMN2 exon 7 inclusion.

Conclusion We identified a novel mechanism that contributes to mis-splicing of SMN2 exon 7.

  • gene expression regulation
  • molecular biology

Data availability statement

Data are available upon reasonable request.

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