An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects

Abstract

A significant proportion of disease-causing mutations affect precursor-mRNA splicing, inducing skipping of the exon from the mature transcript. Using F9 exon 5, CFTR exon 12 and SMN2 exon 7 models, we characterized natural mutations associated to exon skipping in Haemophilia B, cystic fibrosis and spinal muscular atrophy (SMA), respectively, and the therapeutic splicing rescue by using U1 small nuclear RNA (snRNA). In minigene expression systems, loading of U1 snRNA by complementarity to the normal or mutated donor splice sites (5'ss) corrected the exon skipping caused by mutations at the polypyrimidine tract of the acceptor splice site, at the consensus 5'ss or at exonic regulatory elements. To improve specificity and reduce potential off-target effects, we developed U1 snRNA variants targeting non-conserved intronic sequences downstream of the 5'ss. For each gene system, we identified an exon-specific U1 snRNA (ExSpeU1) able to rescue splicing impaired by the different types of mutations. Through splicing-competent cDNA constructs, we demonstrated that the ExSpeU1-mediated splicing correction of several F9 mutations results in complete restoration of secreted functional factor IX levels. Furthermore, two ExSpeU1s for SMA improved SMN exon 7 splicing in the chromosomal context of normal cells. We propose ExSpeU1s as a novel therapeutic strategy to correct, in several human disorders, different types of splicing mutations associated with defective exon definition.

Figure 1.

Effect of natural mutations on F9 exon 5 and CFTR exon 12 pre-mRNA splicing. (A and C) Schematic representation of the central region of the pFIX exon 5 and pCFex12 minigenes, respectively. Exonic sequences are boxed, introns are lines and the arrows represent primers for PCR amplification. The position of genomic variants relative to the splice sites is indicated. Exonic and intronic sequences are in upper and lower case, respectively. (B) Analysis of pFIX exon 5-spliced transcripts. HeLa cells were transfected with pFIXex5 wt or mutant minigenes and splicing pattern evaluated by RT–PCR. Amplified products were resolved on a 2% agarose gel. The identity of the bands is indicated on the right of the panel. M is the molecular 1 Kb marker. Lower panel shows the quantification of the percentage exon 5 inclusion (mean ± SD of three independent experiments done in duplicate). (D) Analysis of pCF exon 12 spliced transcripts. HeLa cells were transfected with pFIX ex12 wt or mutant minigenes and splicing pattern evaluated by RT–PCR. Amplified products were resolved on a 2% agarose gel. Lower panel shows the quantification of the percentage exon 12 inclusion (mean ± SD of three independent experiments done in duplicate).

Figure 2.

Effect of increased complementarity of U1 snRNAs to normal and mutated donor splice site on pre-mRNA splicing. (A) Effect of complementary U1 snRNA to the F9 exon 5 5′ss. The upper panel shows the analysis of spliced transcripts. Minigenes were transfected in HeLa cells with the empty vector (lanes 1, 4, 6, 8, 10, 12, 14) or with modified U1s (lanes 3, 5, 7, 9, 11, 13, 15) and splicing pattern evaluated by RT–PCR. Amplified products were resolved on a 2% agarose gel. The identity of the U1 snRNAs is shown in Supplementary Material, Figure S2. Lower panel is the quantification of exon 5 splicing pattern after co-transfection with modified U1 snRNAs. Data are expressed as means ± SD of three independent experiments done in duplicate. (B) Effect of complementary U1 snRNAs to the CFTR exon 12 5′ss. Minigenes were transfected in HeLa cells with the empty vector (lanes 4, 6, 8) or with modified U1s (lanes 3, 5, 7, 9) and the splicing pattern evaluated by RT–PCR. Amplified products were resolved on a 2% agarose gel. The identity of the U1 snRNAs is shown in Supplementary Material, Figure S2. Lower panel is the quantification of exon 12 splicing pattern. Data are expressed as means ± SD of three independent experiments done in duplicate.

Figure 3.

Identification of ExSpeU1s in F9 exon 5 and CFTR exon 12. (A and C) Schematic representation of binding sites of the ExSpeU1s in F9 exon 5 and CFTR exon 12, respectively. Donor site and downstream intronic region are shown and exonic and intronic sequences are in upper and lower case, respectively. Lines correspond to the target-binding sequences of ExSpeU1s U1 snRNAs on the nascent pre-mRNA. (B) Analysis of F9 exon 5-spliced transcripts. The FIXex5-2C mutant minigene was transfected in HeLa cells alone (lane 1), with the empty vector (lane 2), or with plasmids encoding for the different ExSpeU1s. The pattern of splicing was evaluated by RT–PCR and amplified products were resolved on a 2% agarose gel. Lower panel is the quantification of exon 5 splicing pattern. (D) Analysis of CFTR exon 12-spliced transcripts. The mutant ex12 + 3G minigene was transfected in HeLa cells alone (lane 1), with the empty vector (lane 2), or with plasmids encoding for the ExSpeU1s. The pattern of splicing was evaluated by RT–PCR and amplified products were resolved on a 2% agarose gel. Lower panel is the quantification of exon 5 splicing pattern.

Figure 4.

Identification of ExSpeU1s in SMN2 exon 7. (A) Schematic representation of binding sites of the ExSpeU1s in SMN2 exon 7. Donor site and downstream intronic region are shown and exonic and intronic sequences are in upper and lower case, respectively. Lines correspond to the target-binding sequences of ExSpeU1s on the nascent pre-mRNA. (B) Analysis of SMN2 exon 7 spliced transcripts. The pCI-SMN2 minigene was transfected in HeLa cells alone (lane 1), with the U1wt (lane 2), or with plasmids encoding for ExSpeU1s. The pattern of splicing was evaluated by RT–PCR and amplified products were resolved on a 2% agarose gel. Lower panel is the quantification of SMN2 exon 7 splicing pattern. Data are the means ± SD of three experiments done in duplicate.

Figure 5.

SMN exon 7 splicing correction mediated by sm17 and sm21 ExSpeU1s in normal cells. The upper panel shows a representative experiment in which the fluorescently labelled RT–PCR amplified fragments were separated on denaturing capillary electrophoresis. HEK393 cells were transfected with the indicated ExSpeU1s and amplified fragments were digested with DdeI to obtain SMN1 and SMN2 exon 7 inclusion (FL) and exclusion (Δ7) fragments. The size of the fragments is indicated. Lower panel shows the percentage of SMN1 and SMN2 exon 7 inclusion in ExSpeU1-treated and -untreated cells and data are expressed as means ± SD of three independent experiments done in duplicate. Transfection efficiency in the different experiments was 75–85%.

Figure 6.

Splicing correction mediated by fix9 and cf11 U1 ExSpeU1s on natural mutations. (A) HeLa cells were transfected with the indicated FIX exon 5 mutant minigenes alone (even lanes) or along with fix9 U1 ExSpeU1 (odd lanes). Splicing pattern was evaluated by RT–PCR and amplified products were resolved on a 2% agarose gel. Ms is the molecular 1 Kb plus marker. Lower panel shows the quantification of exon 5 splicing pattern. Percentage of exon inclusion is expressed as means ± SD of three experiments done in duplicate. (B) HeLa cells were transfected with the indicated CFTR exon 12 mutant minigenes alone (even lanes) or along with cf11 ExSpeU1 (odd lanes). Splicing pattern was evaluated by RT–PCR and amplified products were resolved on a 2% agarose gel. Lower panel shows the quantification of exon 12 splicing pattern. Percentage of exon inclusion is expressed as means ± SD of three experiments done in duplicate.

Figure 7.

Rescue of splicing and FIX protein function by ExSpeU1. (A) Schematic representation of the pBsK-FIX minigene. Exonic sequences are boxed, introns are lines, the arrows represent primers for PCR amplification and the dotted lines indicate the two possible exon 5 alternative splicing events. The position of the ATG and TAA codons is indicated. The minigene is not drawn in scale. (B) Analysis of pBsK-FIX exon 5 spliced transcripts. pBsK-FIX exon 5 normal and mutant minigenes were transfected in BHK cells alone or with the indicated ExSpeU1s. The splicing pattern was evaluated by RT–PCR and amplified products were resolved on a 2% agarose gel. M is the molecular 1 Kb marker. Lower panel shows the quantification of the percentage exon 5 inclusion. Data are expressed as means ± SD of three independent experiments done in duplicate. (C) Western blotting of FIX in the BHK conditioned medium. PNP is pooled normal plasma. (D) FIX coagulant activity in the BHK conditioned medium. Data are expressed as means ± SD of three experiments done in duplicate.