Journal: Heliyon

Article Title: RNA binding proteins PTBP1 and HNRNPL regulate CFTR mRNA decay

doi: 10.1016/j.heliyon.2023.e22281

Figure Lengend Snippet: CFTR mRNA abundance in 16HBEs after knockdown (KD) of PTBP1 or HNRNPL. (A) qPCR data quantifying PTBP1 mRNA abundance in WT, G542X, or W1282X 16HBEs after transfection with a scrambled control siRNA (−) or a PTBP1 KD siRNA (+). (B) qPCR data quantifying HNRNPL mRNA abundance in WT, G542X, or W1282X 16HBEs after transfection with a scrambled control siRNA (−) or an HNRNPL KD siRNA (+). (C) qPCR data quantifying CFTR mRNA abundance in WT, G542X, or W1282X 16HBEs after transfection with a scrambled control siRNA (−) or a PTBP1 KD siRNA (+). (D) qPCR data quantifying CFTR mRNA abundance in WT, G542X, or W1282X 16HBEs after transfection with a scrambled control siRNA (−) or a HNRNPL KD siRNA (+). For panels A–D, each column represents the mean ± SD of changes in expression; each symbol represents an individual data point normalized to KRT18 or PPIA from at least three independent experiments (n = 36–54). Data in panels A & B are expressed relative to the scrambled control for each cell line = 100 %. Data in panels C & D are expressed relative to the scrambled control for the WT cell line = 100 %. Statistical analysis was performed for panels A & B using the Welch's unpaired t -test. In panels C & D, statistical analysis was performed using one-way ANOVA. **** indicates p < 0.0001 when comparing the control with an experimental cohort.

Article Snippet: Generation of 16HBEs stably expressing the PTBP1 or HNRNPL isoforms: Isoforms of PTBP1 and HNRNPL carrying a C-terminal hemagglutinin (HA) epitope tag from the pcDNA3.1(+) vector were purchased from GenScript Biotech Corporation.

Techniques: Knockdown, Transfection, Control, Expressing

Journal: Heliyon

Article Title: RNA binding proteins PTBP1 and HNRNPL regulate CFTR mRNA decay

doi: 10.1016/j.heliyon.2023.e22281

Figure Lengend Snippet: CFTR mRNA decay rates in 16HBEs after knockdown (KD) of PTBP1 or HNRNPL. 16HBEs expressing WT or G542X CFTR were transfected with 90 pmol of a scrambled, control siRNA or siRNAs to knockdown PTBP1 or HNRNPL. 48 h after siRNA transfection, 10 μg/mL of actinomycin D was added to the media and RNA was harvested at the indicated times and subjected to RT-qPCR using equal cDNA amounts to quantify the amount of: (A) CFTR or (B) KRT18 remaining after actinomycin D addition relative to the control at time = 0. The data shown are the mean ± SD of the percent of mRNA remaining at each time point, where each experiment was performed twice in quadruplicate (n = 8). (C) Linear regression was performed using semi-log graphs to determine the rate (slope) and half-life of each decay curve, which were tested to determine whether they differed among the cohorts. In the legend, the p values in black font indicate values compared to the WT control; the p values in orange font indicate values compared to the G542X control. p values < 0.05 indicate significant differences between slopes. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Generation of 16HBEs stably expressing the PTBP1 or HNRNPL isoforms: Isoforms of PTBP1 and HNRNPL carrying a C-terminal hemagglutinin (HA) epitope tag from the pcDNA3.1(+) vector were purchased from GenScript Biotech Corporation.

Techniques: Knockdown, Expressing, Transfection, Control, Quantitative RT-PCR

Journal: Heliyon

Article Title: RNA binding proteins PTBP1 and HNRNPL regulate CFTR mRNA decay

doi: 10.1016/j.heliyon.2023.e22281

Figure Lengend Snippet: CFTR mRNA abundance in 16HBEs exogenously expressing PTBP1 and HNRNPL isoforms. (A) Western blots of PTBP1 and HNRNPL isoforms in WT, G542X, or W1282X 16HBEs using an antibody to the C-terminal HA epitope tag. Full, non-adjusted blots are shown in . (B) qPCR data quantifying CFTR mRNA abundance in WT, G542X, and W1282X 16HBEs stably expressing PTBP1 and HNRNPL variants. The data is expressed relative to the scrambled control for each cell line = 1. Each column represents the mean ± SD of changes in expression; each symbol represents an individual data point normalized to KRT18 or PPIA from at two independent experiments performed in quadruplicate (n = 16). Statistical analysis was performed using the Welch's unpaired t -test; **** indicates p < 0.0001 when comparing the control with the experimental cohort for each cell line.

Article Snippet: Generation of 16HBEs stably expressing the PTBP1 or HNRNPL isoforms: Isoforms of PTBP1 and HNRNPL carrying a C-terminal hemagglutinin (HA) epitope tag from the pcDNA3.1(+) vector were purchased from GenScript Biotech Corporation.

Techniques: Expressing, Western Blot, Stable Transfection, Control

Journal: Heliyon

Article Title: RNA binding proteins PTBP1 and HNRNPL regulate CFTR mRNA decay

doi: 10.1016/j.heliyon.2023.e22281

Figure Lengend Snippet: Model of how PTBP1 and HNRNPL may regulate NMD of CFTR mRNAs. (A) PTBP1, but not HNRNPL, regulates WT CFTR mRNA abundance, likely by protecting these transcripts from EJC-independent NMD that is mediated by the accumulation of UPF1 on the long 3′ UTR. (B) Both PTBP1 and HNRNPL regulate PTC-containing CFTR mRNA abundance, suggesting that PTC-containing CFTR transcripts may be subject to both EJC- and long 3′ UTR-mediated NMD. We speculate that PTBP1 may protect CFTR mRNAs from 3′ UTR mediated NMD, while HNRNPL may protect CFTR mRNAs from EJC-mediated NMD that likely recruits different NMD-associated factors based on the location of the PTC. CU and CA represent binding motifs for PTBP1 and HNRNPL, respectively.

Article Snippet: Generation of 16HBEs stably expressing the PTBP1 or HNRNPL isoforms: Isoforms of PTBP1 and HNRNPL carrying a C-terminal hemagglutinin (HA) epitope tag from the pcDNA3.1(+) vector were purchased from GenScript Biotech Corporation.

Techniques: Binding Assay