hnRNP A1 induces aberrant CFTR exon 9 splicing via a newly discovered ESS element

Abstract

RNA-protein interactions play a key role in the aberrant splicing of CFTR exon 9. Exon 9 skipping leads to the production of a nonfunctional chloride channel associated with severe forms of cystic fibrosis. The missplicing depends on TDP-43 binding to an extended UG-rich binding site upstream of CFTR exon 9 3' splicing site (3'ss) and is associated with concomitant hnRNP A1 recruitment. Although TDP-43 is the dominant inhibitor of exon 9 inclusion, the role of hnRNP A1, a protein with two RNA recognition motifs, remained unclear. In this work, we have studied the interaction between hnRNP A1 and the CFTR pre-mRNA using NMR spectroscopy and Isothermal Titration Calorimetry. The affinities are submicromolar, and Isothermal Titration Calorimetry data suggest complexes with a 1:1 stoichiometry. NMR titrations reveal that hnRNP A1 interacts with model CTFR 3'ss sequences in a fast exchange regime at the NMR timescale. Splicing assays finally show that this hnRNP A1 binding site represents a previously unknown exonic splicing silencer element. Together, our results shed light on the mechanism of aberrant CFTR exon 9 splicing.

Figure 1.. Aberrant splicing of CFTR exon 9.

(A) Recognition of the CFTR 3′ss by TDP-43 and hnRNP A1. The relevant RNA sequences in intron 8 and exon 9 are displayed. TDP-43 binds the elongated UG repeat and recruits hnRNP A1 via their C-terminal glycine-rich domains. Access of SF1/BBP and U2AF35/65 to the canonical binding sites (BPS, polypyrimidine tract, and 3′ss AG) at the 3′ss is impaired. The pre-mRNA interaction site of hnRNP A1 is unknown. (B) Schematic domain representation of human TDP-43 RBD and human hnRNP A1. Domain boundaries of each domain are indicated according to the full-length protein sequences (NP_031401 and NP_112420.1).

Figure 2.. Minigene splicing assay identifies a novel ESS motif in CFTR exon 9.

(A) Schematic representation of the CFTR exon 9 minigene construct used in the transfection experiments. Lines represent introns, and boxes represent exons. The intronic TG11-T5 TDP-43 binding site upstream of exon 9 is also indicated. Dashed lines indicate alternative pre-mRNA processing of the minigene, and the two major splice forms are shown on the right. (B) Binding sites of TDP-43, and hnRNP A1 and A2 predicted by DeepCLIP (https://deepclip-web.compbio.sdu.dk) (Gronning et al, 2020) using pretrained models from RNAcompete data sets (Ray et al, 2013). The sequence is displayed below, and the conserved AG at the 3′ss is highlighted in bold. The predicted bipartite binding site for hnRNP A1 and A1, a potential ESS motif, is shaded. (C) CFTR exon 9 inclusion upon mutation of the ESS motif in exon 9. The TG11-T5 CFTR exon 9 reporter minigenes (wt and mutants mut1, mut3, mut4, mut7) were transfected in HeLa cells. Progressive mutations of the ESS motif lead to full inclusion of exon 9.

Figure 3.. Overexpression of hnRNP A1 protein in the presence of the CFTR exon 9, mut3, and mut4 minigenes.

(A) CFTR exon 9 reporter minigenes (wt and mutants mut3 and mut4) were transfected in HeLa cells with and without hnRNP A1 overexpression. Band sizes are indicated and correspond to inclusion of CFTR exon 9 (upper band) and exclusion (lower band). (B) Summarized results from three biological replicates (four individual transfection experiments); one-way ANOVA was performed, and data are shown as the mean ± SD.

Figure 4.. Effects of hnRNP A1 and A2 knockdown on CFTR exon 9 inclusion, using the pTB-CFTR exon 9 C155T plasmid (Pagani et al, 2000).

(A) Two graphs report the level of hnRNP A1 and hnRNP A2 mRNA knockdown with siRNAs, siA1 and siA2, respectively, or control siRNA (siLUC) as determined by RT–qPCR analysis. Three independent experiments were plotted in a column graph, and an unpaired t test was performed using GraphPad software. (B) Representative gel obtained using QIAxcel High Resolution Kit (QIAGEN) showing the inclusion of CFTR exon 9 (upper band) and exclusion (lower band) after treatment with hnRNP A1/A2 siRNA (siA1A2) and control siRNA (siLUC). (C) Quantification of CFTR exon 9 inclusion from three independent experiments. The quantification was performed using QIAxcel High Resolution Kit software (QIAGEN), and the single values were plotted in a column graph: an unpaired t test was performed using GraphPad software.

Figure 5.. hnRNP A1 binds the novel bipartite ESS motif in CFTR exon 9.

(A) Affinity of the hnRNP A1 tandem RRMs for the bipartite ESS motif was determined by Isothermal Titration Calorimetry (ITC). The determined K_d value is indicated with SD, as well as ΔH and ΔS. (B) Affinity of hnRNP A1 RRM1 for the bipartite ESS motif was determined by ITC. Two copies of hnRNP A1 RRM1 bind to the bipartite ESS motif. The determined K_d value is indicated with SD. (C) Affinity of hnRNP A1 RRM2 for the bipartite ESS motif was determined by ITC. Two copies of hnRNP A1 RRM2 bind to the bipartite ESS motif. The determined K_d value is indicated with SD. (D) Affinity of the hnRNP A1 tandem RRMs for the bipartite wt (top) and mut4 (bottom) ESS motif was measured by fluorescence anisotropy. The determined K_d value represents the average of three individual measurements and is indicated with SD.

Figure 6.. Both hnRNP A1 RRM1 and RRM2 bind the individual segments of the ESS motif of CFTR exon 9.

(A, B) Affinity of hnRNP A1 RRM1 for the individual segments of the ESS motif was determined by Isothermal Titration Calorimetry. 1:1 complexes are formed with the individual segments of the ESS motif. The determined K_d values are indicated with SD. (C, D) Affinity of hnRNP A1 RRM2 for the individual segments of the ESS motif was determined by Isothermal Titration Calorimetry. 1:1 complexes are formed with the individual segments of the ESS motif. The determined K_d values are indicated with SD.

Figure 7.. NMR titration of hnRNP A1 RRM1 reveals specific interactions with the individual segments of the ESS motif.

(A, C) Superimposition of ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled hnRNP A1 RRM1 in the free (blue) and short ESS motif-bound form (red). The amide cross-peaks shifted upon interaction are labeled with amino acid type and residue number. (B, D) Combined chemical shift perturbations Δδ = [(δH^N)² + (δN/6.51)²]^1/2 of hnRNP A1 RRM1 amide resonances upon short ESS motif binding versus the hnRNP A1 RRM1 amino acid sequence.

Figure 8.. NMR titration of hnRNP A1 RRM2 reveals specific interactions with the individual segments of the ESS motif.

(A, C) Superimposition of ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled hnRNP A1 RRM2 in the free (blue) and short ESS motif-bound form (red). The amide cross-peaks shifted upon interaction are labeled with amino acid type and residue number. (B, D) Combined chemical shift perturbations Δδ = [(δH^N)² + (δN/6.51)²]^1/2 of hnRNP A1 RRM2 amide resonances upon short ESS motif binding versus the hnRNP A1 RRM2 amino acid sequence.

Figure 9.. hnRNP A1 tandem RRMs display a continuous binding site for the novel CFTR exon 9 ESS motif.

The CSPs from the titration experiments of the individual hnRNP A1 RRMs and the short segments of the ESS motif are displayed on the structure of the hnRNP A1 tandem RRMs (PDB ID: 2lyv). RRM1 is colored in light gray and RRM2 in dark gray. The CSPs are indicated on the ribbon structure in salmon, and the sequence of the corresponding ESS segments is displayed on top.