Decreased mRNA and protein stability of W1282X limits response to modulator therapy

Abstract

Background: Cell-based studies have shown that W1282X generates a truncated protein that can be functionally augmented by modulators. However, modulator treatment of primary cells from individuals who carry two copies of W1282X generates no functional CFTR. To understand the lack of response to modulators, we investigated the effect of W1282X on CFTR RNA transcript levels.

Methods: qRT-PCR and RNA-seq were performed on primary nasal epithelial (NE) cells of a previously studied individual who is homozygous for W1282X, her carrier parents and control individuals without nonsense variants in CFTR.

Results: CFTR RNA bearing W1282X in NE cells shows a steady-state level of 4.2 ± 0.9% of wild-type (WT) CFTR RNA in the mother and 12.4 ± 1.3% in the father. NMDI14, an inhibitor of nonsense-mediated mRNA decay (NMD), restored W1282X mRNA to almost 50% of WT levels in the parental NE cells. RNA-seq of the NE cells homozygous for W1282X showed that CFTR transcript level was reduced to 1.7% of WT (p-value: 4.6e-3). Negligible truncated CFTR protein was generated by Flp-In 293 cells stably expressing the W1282X EMG even though CFTR transcript was well above levels observed in the parents and proband. Finally, we demonstrated that NMD inhibition improved the stability and response to correctors of W1282X-CFTR protein expressed in the Flp-In-293 cells.

Conclusion: These results show that W1282X can cause substantial degradation of CFTR mRNA that has to be addressed before efforts aimed at augmenting CFTR protein function can be effective.

Figure 1. Assessment of CFTR mRNA bearing W1282X in the primary nasal epithelial cells of healthy heterozygous carriers of W1282X.

(A) Pedigree of family with W1282X CFTR mutation demonstrating segregation consistent with autosomal recessive inheritance of CF. (B) Electropherograms from Sanger sequencing of genomic DNA confirmed the segregation of W1282X CFTR mutation as indicated in the pedigree. Polymerase chain reaction (PCR) was performed on 50 ng genomic DNA using CFTR specific primers (10 μM each) from introns flanking exon 23. The W1282X mutation is caused by the replacement of a “G” nucleotide at 3846 by a “A” nucleotide (compare heterozygous parents and homozygous proband tracings). (C) Reverse transcription-PCR analysis of W1282X CFTR. RT-PCR was performed on cDNAs using CFTR-specific primers from exon 22 and exon 24. Following electrophoresis on a 2% agarose gel, a single amplification product was visualized. The molecular weight of the amplification product matched the expected size of a product (417 base pairs, based on the position of RT-PCR primers in the 5’-exon 22 and 3’-exon 24). TATA box binding protein (TBP) was amplified as control. No-RT and water lanes are negative controls. (D) Direct sequencing of the RT-PCR product to assess differential expression of W1282X mRNA in heterozygous parents. Top, electropherograms obtained from Sanger sequencing (compare area under the peak for “nucleotide A” and “nucleotide G” indicated by arrows corresponding to W1282X and WT alleles respectively. Bottom, Graphs showing relative expression of W1282X and WT evaluated by pyrosequencing. Assay was designed such that exon 23 with upstream and downstream flanking exons was amplified from the corresponding cDNA preparations. Sequencing primer yielded relative abundance of alternate alleles at the W1282 codon (n=3, Mean±SEM).

Figure 2. Transcriptome analysis of the primary nasal epithelial cells of W1282X proband, carrier parents, and controls.

(A) Diagram of methodology used to generate and analyze RNA-seq data. (B) Volcano plot (x-axis: log10(fold change), y-axis: −log10(p-value)) comparing global mRNA expression of autosomal genes with gene expression greater than 0.1 FPKM between two controls groups (individuals do not contain nonsense variants in CFTR); obtained and sequenced in house vs. obtained from SRA from a study conducted at George Washington (GW) University (study accession numbers SRR1528464 and SRR1528468) (14,324 genes) (C) Volcano plot of differential gene expression between the proband and control (n=14,323 genes). (D) Volcano plot of differential gene expression between the carrier mother and control (n=3 genes). (E) Volcano plot of differential gene expression between carrier father and control (n=18,733 genes).

Figure 3. Evaluation of W1282X mRNA stability in primary nasal epithelial cells of W1282X carrier parents.

(A) Left, Schematic illustration showing premature termination codon (PTC), e.g. W1282X, recognized by components of nonsense mediated decay (NMD). NMDI14 inhibits interaction of UPF1 (grey) and SMG7 (red) proteins that assemble at the PTC, as shown previously (23). Right, Schematic illustration of well-differentiated primary nasal epithelial (NE) cells of the carrier parents that express W1282X and WT CFTR RNA. NE cells were conditionally reprogrammed (CR) using Rho-kinase inhibitor. CRNE cells were transferred on snapwells and exposed to air at the apical side to establish air-liquid interface (ALI) culture. Four-week old ALI culture was used to assess mRNA stability. (B) Assessment of W1282X mRNA stability in ALI cultures of both carrier parents. Cells were treated with vehicle (DMSO 0.1%) or NMDI14 (5 μM) for 12 hours. Actinomycin D (2 μM) was added to terminate mRNA synthesis. RNA was collected at time points indicated on the graph after addition of actinomycin D. Relative expression of W1282X mRNA was assessed by pyrosequencing. Data are Mean±SEM, n=2 from each parent.

Figure 4. Assessment of mRNA stability and protein production in cell line model expressing an expression mini-gene (EMG) bearing W1282X CFTR

(A) A schematic of CFTR-Expression Minigene with abridged introns 21, 22, 23 and 24 (EMG-i21-i24) constructed in pcDNA5FRT plasmid. CFTR exons are shown in boxes and abridged introns in dashed lines. Location of W1282X variant in the plasmid DNA is indicated. EMGi21-i24 results in normal splicing. Both WT and W1282X EMG splice normally. CFTR mRNA splicing patterns of the total RNA extracted from Flp-In-293 cells transiently transfected with E831X-EMG. (B) RT-qPCR showing relative steady state levels of CFTR transcript in Flp-In-293 stable cells expressing wild-type EMG or W1282X EMG. Values were normalized to B2M. Mean ± SEM (n=3) measured in triplicates. (C) CFTR mRNA decay in Flp-In-293 cells stably expressing wild type EMG, and W1282X EMG. Actinomycin D (2 μM) was added at time 0 to induce transcriptional shut-down. Cells were collected at the indicated time points. Levels of the CFTR mRNAs were assessed by RT-qPCR, normalized to B2M mRNA and displayed as a percentage of the levels at t=0. Mean ± SEM (n=3). (D) Steady state levels of CFTR protein in Flp-In-293 stable cells expressing wild-type EMG or W1282X EMG. 40 μg of total cell lysates were electrophoresed and Immunoblot (IB) was probed with anti-CFTR antibody, 596 (CFFT). Lysates from cells expressing intronless WT CFTR or F508del served as positive controls, and non-transfected Flp-in 293 cells as negative control. Na⁺K⁺ATPase was used as loading control. (E) Left, shows immunoblot of W1282X CFTR-EMGi21–24, stably expressed in Flp-In-293 cells in response to correctors (lumacaftor 3 μM, tezacafor 3 μM, and their combination) either alone or in combination with NMD inhibitor (NMDI14 5 μM). Both correctors, and NMDI14 treatments were given for 24 h. Arrows represent immature core-glycosylated and mature complex-glycosylated truncated W1282X protein generated from W1282X-EMG in Flp-In-293 stable cells. Lysates collected from Flp-In-293 stable cells expressing intronless WT CFTR or F508del served as positive controls, and non-transfected Flp-in-293 cells as negative control. Na⁺K⁺ATPase was used as loading control. Right, shows the quantitative assessment through densitometry findings of three different exposures of that experiment (Mean±SEM).