Nuclear factor TDP-43 and SR proteins promote in vitro and in vivo CFTR exon 9 skipping

Abstract

Alternative splicing of human cystic fibrosis transmembrane conductance regulator (CFTR) exon 9 is regulated by a combination of cis-acting elements distributed through the exon and both flanking introns (IVS8 and IVS9). Several studies have identified in the IVS8 intron 3' splice site a regulatory element that is composed of a polymorphic (TG)m(T)n repeated sequence. At present, no cellular factors have been identified that recognize this element. We have identified TDP-43, a nuclear protein not previously described to bind RNA, as the factor binding specifically to the (TG)m sequence. Transient TDP-43 overexpression in Hep3B cells results in an increase in exon 9 skipping. This effect is more pronounced with concomitant overexpression of SR proteins. Antisense inhibition of endogenous TDP-43 expression results in increased inclusion of exon 9, providing a new therapeutic target to correct aberrant splicing of exon 9 in CF patients. The clinical and biological relevance of this finding in vivo is demonstrated by our characterization of a CF patient carrying a TG10T9(DeltaF508)/TG13T3(wt) genotype leading to a disease-causing high proportion of exon 9 skipping.

Fig. 1. (A) A schematic representation of the intronic and exonic elements that affect the splicing of CFTR exon 9: the (TG)m and (T)n polymorphic regions in IVS8, the intronic splicing silencer (ISS) in IVS9, and the exonic enhancer (E) and silencer (S) sequences. (B) A schematic representation of the plasmids used: wild type (11ug/7u) and mutants selectively deleted of the (TG)m and/or (T)n repeats (Δug/Δu), (11ug/Δu) and (Δug/7u). (C) A UV cross-linking assay using HeLa nuclear extract with uniformly labeled RNA of all four constructs linearized with HindIII. The arrows indicate the position of the 50–52 kDa complex.

Fig. 2. (A) A competition analysis following addition of cold 11ug/7u, Δug/Δu, 11ug/Δu and Δug/7u RNAs to labeled (11ug/7u) RNA in the presence of HeLa nuclear extract. The molar ratios of cold/labeled RNA were 2, 5 and 10. (B) A schematic representation of the human (11ug/7u) and mouse (mEx9) constructs. (C) A competition analysis using labeled 11ug/7u RNA incubated with HeLa nuclear extracts following addition of cold RNAs: 11ug/7u RNA and two RNAs synthesized by cutting mEx9 with EcoRI (mEx9EcoRI) and with HindIII (mEx9HindIII). The molar ratios of cold/labeled RNA were 2, 5 and 10. The arrows indicate the 50–52 kDa complex.

Fig. 3. (A) The effects of two short competitor RNAs, (ug)₁₂ and (ucuu)₃, in a UV cross-linking assay in the presence of HeLa nuclear extract and labeled 11ug/7u RNA. The molar ratios of cold/labeled RNA were 3, 8 and 17. (B) The results of labeling the short (ug)₁₂ RNA and performing UV cross-linking in the presence of HeLa nuclear extract and cold (ug)₁₂ and (ucuuu)₃ RNAs. The molar ratios of cold/labeled RNA were 2 and 5. The arrows indicate the 50–52 kDa complex.

Fig. 4. (A) The results (Coomassie Blue staining) of a pull-down assay using adipic acid dehydrazide beads derivatized with (ug)₁₂ and (ucuu)₃ RNAs following incubation with HeLa nuclear extract. In the lane from the (ug)₁₂-derivatized beads the arrow indicates the 43 kDa protein band that is absent in the lane from the (ucuu)₃-derivatized beads. The arrows on the right indicate the 57 kDa doublet that is present in the (ucuu)₃ lane as opposed to the (ug)₁₂ lane. (B) The full amino acid sequence of TDP-43 with the open box corresponding to the sequenced peptide from the excised 43 kDa band. Bold and underlined sequences highlight the two RRM consensus motifs. (C) (Left panel) The purified recombinant proteins and their reactivity with labeled (ug)₁₂ RNA in a UV cross-linking assay (right panel). (D) The reactivity of the GST–TDP-43 recombinant protein with labeled 11ug/7u, Δug/7u, 11ug/Δu and Δug/Δu RNAs.

Fig. 5. (A) (Upper panel) The reactivity of labeled 11ug/7u RNA in the presence of 18 µg of HeLa nuclear extract (NE) and 18 µg of TDP-43 immunodepleted nuclear extract (NE-TDP-43). (Lower panel) A western blot assay demonstrating that immunodepletion of TDP-43 has occurred. (B) An immunoprecipitation experiment using anti-TDP-43 sera (and its pre-immune sera as control) on labeled 11ug/7u and Δug/Δu RNAs UV cross-linked with 18 µg of nuclear extract. (C) An immunoprecipitation following UV cross-linking of labeled 11ug/7u RNA with increasing quantities of nuclear extract. The arrow indicates the 50–52 kDa immunoprecipitated product whilst the asterisk indicates the second immunoprecipitated band. (D) The effect of UV cross-linking labeled 11ug/7u RNA with GST–TDP-43 alone (50 ng), with nuclear extract alone (18 µg) and nuclear extract mixed with increasing quantities of GST–TDP-43 (10, 25 and 50 ng, respectively). The 50–52 kDa complex and GST–TDP-43 protein are indicated by arrows. (E) A schematic diagram (top) of the recombinant GST–TDP-43(101–261), its expression and purification (left panel), and its reactivity with synthetic 5′ end-labeled (ug)₁₂ RNA following UV cross-linking with (+) and without (–) RNase treatment (right panel). In the third lane (–/+) these two were mixed and loaded together in the same lane. Only 10% of the untreated sample was loaded in the (–) and (–/+) lanes. The lower amount of labeled material in the (+) lane is due to loss of the labeled 5′ end of the synthetic (ug)₁₂ following RNase digestion.

Fig. 6. (A) A schematic representation of the hybrid minigene, hCF-(TG)m(T)n. The minimal α-globin promoter and SV40 enhancer are indicated by a small arrow at the 5′ end, the polymorphic locus (TG)m(T)n by a gray circle, and the α-globin, fibronectin EDB and human CFTR exons by black, shaded and white boxes, respectively. The primers used in the RT–PCR assay are indicated by the superimposed arrows. (B) Left panel, the expression of selected minigene variants in the presence of plasmids overexpressing either TDP-43 or SF2/ASF. Exon 9 positive (+) and negative (–) bands are indicated. The arrow indicates an aberrant splicing product originating from a cryptic 3′ splice site. The percentage of exon exclusion for each construct either alone or in the presence of SF2/ASF (500 ng), TDP-43 (3 µg) or both, is reported in the lower graph. Right panel, the effect of TDP-43 and SF2/ASF overexpression on the fibronectin EDA exon. (C) Left panel, a dose–response curve of exon 9 exclusion in the presence of increasing amounts of TDP-43 (0.5–5 µg transfected plasmid) on the TG13T5 minigene. Mean values from four independent transfection experiments performed as duplicates are shown with standard errors (right panel). The asterisks indicate statistical significance (P <0.05). M, molecular weight markers (1 kb).

Fig. 7. Antisense inhibition of TDP-43 in Hep3B cells transfected with the TG13T5 minigene. (A) A schematic diagram of four PS-oligodeoxy nucleotides (TIO7, TIO86, TIO155 and TIO1318) used. Hep3B cells were co-transfected with 3 µg of TG13T5 minigene and each PS- or a control oligo (FN56) at a final concentration of 1 µM (B, left panel). A TG13T3 control was also included (pRc/CMV). Exon 9 inclusion levels are reported (right panel). (C) A dose–response curve with oligo TIO1318 ranging from 0.1 to 5 µM (upper panel) together with a western blot of endogenous TDP-43 levels (lower panel).

Fig. 8. (A) Northern blot analysis of TDP-43 (upper panels), SF2/ASF (middle panels) and GAPDH (lower panels) performed on mRNA extracted from 16 different human tissues. The arrows indicate the major 2.8 kb mRNA species characteristic of TDP-43 and the 3.0 kb mRNA of SF2/ASF. (B) A graphical representation of the normalized TDP-43 mRNA levels (black boxes) and SF2/ASF levels (shaded boxes).

Fig. 9. (A) A family tree and a sequencing analysis of the pancreatic-sufficient CF patient carrying the TG13T3(wt) allele on one chromosome and the TG10T9(ΔF508) configuration on the other chromosome. (B) RT–PCR products spanning exons 8–11 of the CFTR cDNA, obtained from the CF patient compound heterozygous for the TG13T3 mutation and the TG10T9(ΔF508) allele (lane 1) and from a control individual compound heterozygous for the TG11T7 and TG10T7 alleles (lane 2), separated on a 2% agarose gel. The percentage of exon 9 exclusion in each CFTR transcript from the two alleles, as determined after denaturing PAGE separation, is given below. Lane 3, no template; M, 1 kb marker. (C) A semiquantitative analysis of exon 9 skipping. The RT–PCR products from the CF patient (upper profile), a control individual (middle profile), and a negative control (lower profile) were separated on a denaturing polyacrylamide gel using an ALF sequencer. Fluorescence signals were quantified using the Fragment Manager software. Peaks 1 and 3 correspond to the amplified 9(–) and 9(+) fragments from the TG10T9(ΔF508) allele whilst peaks 2 and 4 correspond to amplified 9(–) and 9(+) fragments from non-ΔF508 alleles.