Antisense Oligonucleotide Induction of the hnRNPA1b Isoform Affects Pre-mRNA Splicing of SMN2 in SMA Type I Fibroblasts

Abstract

Spinal muscular atrophy (SMA) is a severe, debilitating neuromuscular condition characterised by loss of motor neurons and progressive muscle wasting. SMA is caused by a loss of expression of SMN1 that encodes the survival motor neuron (SMN) protein necessary for the survival of motor neurons. Restoration of SMN expression through increased inclusion of SMN2 exon 7 is known to ameliorate symptoms in SMA patients. As a consequence, regulation of pre-mRNA splicing of SMN2 could provide a potential molecular therapy for SMA. In this study, we explored if splice switching antisense oligonucleotides could redirect the splicing repressor hnRNPA1 to the hnRNPA1b isoform and restore SMN expression in fibroblasts from a type I SMA patient. Antisense oligonucleotides (AOs) were designed to promote exon 7b retention in the mature mRNA and induce the hnRNPA1b isoform. RT-PCR and western blot analysis were used to assess and monitor the efficiency of different AO combinations. A combination of AOs targeting multiple silencing motifs in hnRNPA1 pre-mRNA led to robust hnRNPA1b induction, which, in turn, significantly increased expression of full-length SMN (FL-SMN) protein. A combination of PMOs targeting the same motifs also strongly induced hnRNPA1b isoform, but surprisingly SMN2 exon 5 skipping was detected, and the PMO cocktail did not lead to a significant increase in expression of FL-SMN protein. We further performed RNA sequencing to assess the genome-wide effects of hnRNPA1b induction. Some 3244 genes were differentially expressed between the hnRNPA1b-induced and untreated SMA fibroblasts, which are functionally enriched in cell cycle and chromosome segregation processes. RT-PCR analysis demonstrated that expression of the master regulator of these enrichment pathways, MYBL2 and FOXM1B, were reduced in response to PMO treatment. These findings suggested that induction of hnRNPA1b can promote SMN protein expression, but not at sufficient levels to be clinically relevant.

Keywords: antisense oligonucleotide; hnRNPA1; phosphorodiamidate morpholino oligomer; spinal muscular atrophy; transcriptome.

Figure 1

Schematic diagram displaying the targeting splice motifs and AO coordinates of ten AOs induced hnRNPA1 exon 7b skipping (AOs1 and 2) and inclusion (AOs3–10).

Figure 2

RT-PCR analysis demonstrated splicing patterns of hnRNPA1, (A) and percentage of exon 7b inclusion in SMA fibroblasts transfected with different 2′O-methyl modified AOs (B).

Figure 3

Gel images and densitometric analysis of RT-PCR of hnRNPA1 (A,B) and SMN (C,D) splicing isoforms in SMA fibroblasts transfected by seven combinations of AO cocktails, as well as densitometric analysis of the percentage of FL-SMN. The indicated concentrations were a dose of combined AOs in the cocktail. The * symbol indicates the p-value of the average percentage of exon inclusion compared to untreated samples (viz ** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001).

Figure 4

Western blot analysis indicates the hnRNPA1 (34 kDa) and the induced isoform of hnRNPA1 proteins at approximately 38 kDa (A), SMN protein at approximately 38 kDa (C) and β-tubulin protein at approximately 50 kDa (C) expressed in SMA fibroblasts that were transfected with selected seven different AO cocktails, as well as densitometric analysis of percentage of hnRNPA1b (B), as compared to SMN protein expression normalised against β-tubulin (D). The * symbol indicates the p-value of the average relative protein expression compared to untreated samples (viz * = p < 0.05, ** = p < 0.01 and *** = p < 0.001).

Figure 5

Gel images of RT-PCR shows hnRNPA1 (827 bp) and hnRNPA1b (983 bp) transcripts (A). Panel (B),(C) shows gel images of different isoforms of SMN2 transcripts missing exon 5 (Δ5), missing exon 7 (Δ7) or missing both exons 5 and 7 (Δ5 + 7) with densitometric analysis of SMN2 transcript. Western blot analysis of hnRNPA1, FL-SMN and β-tubulin expression in SMA fibroblasts transfected with selected seven different PMO cocktails as well as densitometric analysis of percentage of hnRNPA1b compared to SMN protein expression normalised against β-tubulin (D–G). Numbers above each bar graph indicates the average values of percentage of hnRNPA1b isoform (E) and the average fold change of increased SMN protein (G). ** = p < 0.01, *** = p < 0.001.

Figure 6

Differential expression comparing between PMOs9 + 7 + 8 treated SMA fibroblast and untreated SMA fibroblasts. Heatmap of the differentially expressed genes in three replicates of both conditions. (A) Volcano plot of differentially expressed gene list comparing between cells transfected with PMOs9 + 8 + 7 and untreated SMA fibroblasts. (B) Selected genes with absolute log2 fold change greater than 1.5 folds and p-value lesser than 0.05 was analysed for its gene ontology. Top twenty of biological processes that were upregulated (C) or downregulated (D) in PMO treatment group compared to untreated control. Gene set enrichment analysis indicated that gene set regulating cell cycles were enriched at the normalised enrichment score (NES) of −1.8477 and false discovery rate (FDR) q-value of 0.00 (E).

Figure 7

Isoform specific RT-PCR to detect four different isoforms of FOXM1 (A) with the graph displaying band densitometry in the panels (B–E) (viz FOXM1a, FOXM1b, FOXM1c and FOXM1d) showed reduced expression of FOXM1b in cultured SMA fibroblasts treated with the combination of PMOs 9 + 8 + 7, while RT-PCR analysis of MYBL2 (F) was also reduced, as indicated in its band densitometry (G).