Fig 2: Characterization of the CFF-16HBEge in vitro model system.CFF-16HBEge cells with CFTR Y122X, G542X, R1162X, and W1282X nonsense mutations have significantly reduced CFTR expression and function when compared to parental cells. a A schematic displaying CFTR nonsense mutations which were evaluated in this study. b RT-qPCR analysis of CFTR mRNAs in parental and CFF-16HBEge cells with Y122X, G542X, R1162X, or W1282X CFTR nonsense mutations. CFTR mRNA levels were normalized to total RNA levels (n = 3 for each group). The wild-type (WT) CFTR mRNA level in parental cells was set to 1 and is indicated by the dotted red line. c Representative western blot image of CFTR protein expression in parental and CFF-16HBEge cells. Na+/K+-ATPase (NKA) protein was used as a loading control (n = 3 for each group). d Representative short circuit current (Isc) traces of parental and CFF-16HBEge cells obtained using the Ussing Chamber assay. 7 µM benzamil was added to establish baseline current followed by the sequential addition of 20 µM forskolin, 1 µM VX-770, and 20 µM CFTR inhibitor-172 (CFTRinh-172) at times indicated by arrows. e ΔIsc of CFTR-attributed current in parental and CFF-16HBEge cells (n = 4 for parental, n = 5 for CFF-16HBEge-Y122X, n = 3 for CFF-16HBEge-G542X, n = 4 for CFF-16HBEge-R1162X, and n = 5 for CFF-16HBEge-W1282X cells). f A schematic displaying the recognition, tagging, and decay steps of the NMD pathway. Core NMD factors are indicated by the color blue, SMG1-cofactors SMG8 and SMG9 are indicated by the color purple, and branch-specific NMD components are colored red. Data are presented as mean ± SEM from biologically independent samples. Statistical significance was analyzed by one-way ANOVA followed by Dunnett’s multiple-comparison test (***p < 0.001, ****p < 0.0001). Black stars indicate statistical comparisons made between parental cells and CFF-16HBEge cells. Red stars indicate statistical comparisons made between CFF-16HBEge-Y122X and CFF-16HBEge-G542X, -R1162X, and -W1282X cells. See Source Data file for the exact p-values. Source data are provided as a Source Data file.

Fig 3: SMG6-ASO treatment improves translational readthrough outcomes and enhances CFTR function improvement in CFF-16HBEge cells.Cells were treated with SMG1- or SMG6-ASO for 6 days followed by the addition of aminoglycoside geneticin (G418; 100 µM) during the final 2 days of ASO treatments. Representative Isc traces (left) and ΔIsc (right) were obtained from Ussing chamber assays of CFF-16HBEge cells with (a, b) Y122X, (c, d) G542X, (e, f) R1162X, or (g, h) W1282X CFTR nonsense mutations following the indicated treatment conditions. For the Ussing chamber assay, 7 µM benzamil was added to establish baseline currents followed by the sequential addition of 20 µM forskolin, 1 µM VX-770, and 20 µM CFTR inhibitor-172 (CFTRinh-172) at times indicated by arrows to determine CFTR function. n = 3 for each group. Data are presented as mean ± SEM from biologically independent samples. Statistical significance was analyzed by one-way ANOVA followed by Sidak’s multiple-comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). See Source Data file for the exact p-values. Source data are provided as a Source Data file.

Fig 4: SMG1 regulates RMD through the direct phosphorylation of UPF1. (A) Luciferase activity of HeLa cells treated with indicated inhibitors, including Compound 11J (SMG1 inhibitor, 0.3 µM), KU-60019 (ATM inhibitor, 1 µM), VE-821 (ATR inhibitor, 1 µM) and NU7441 (DNA-PK inhibitor, 1 µM), and transfected with indicated luciferase reporter plasmids, together with control plasmid or indicated expression plasmids. (B) HEK293 Tet-off cells were transfected with siRNA specific for SMG1 and cotransfected with pTREtight-Il6 CDS-3' UTR together with the Regnase-1 expression plasmid or control (Mock) plasmid. Total RNAs were prepared after Dox (1 µg/ml) treatment, and Il6 and ß-Actin mRNA levels were determined by Northern blot analysis. (C) Quantification of the autoradiographs in (B), presented as the ratio of Il6 to ß-Actin (n = 3). (D) Luciferase activity of HeLa cells transfected with indicated siRNA and luciferase reporter plasmids, together with control plasmid (Mock) or Regnase-1 expression plasmids. (E) RNA expression levels in HeLa cells transfected with indicated siRNA and stimulated with IL-1ß (10 ng/ml). (F) Western blot analysis of SMG1 in HeLa cells transfected with siRNA specific for SMG1 and reconstituted with siRNA-resistant Flag-SMG R-WT or Flag-SMG1R-D2331A (DA), which is a kinase dead mutant. (G) Luciferase activity of HeLa cells transfected with siRNA specific for SMG1 and reconstituted with siRNA-resistant WT or DA mutant of SMG1 and then transfected with indicated luciferase reporter plasmids, together with control plasmid (Mock) or Regnase-1 expression plasmids. (H) RNA expression levels in HeLa cells transfected with siRNA specific for SMG1, reconstituted with siRNA-resistant WT or DA mutant of SMG1 and then stimulated with IL-1ß (10 ng/ml). See also Supplementary Figure S3.

Fig 5: Interactions between SMG1C and DHX34.(a) SDS–PAGE of purified FLAG-DHX34 and FLAG-SBP-HA-SMG1C used for structural studies. Purified proteins were resolved in a 4–15% SDS–PAGE and stained using Oriole Fluorescent Gel Stain. (b) Pull-down experiment testing the interaction of purified FLAG-DHX34 and FLAG-SBP-HA-SMG1C. Proteins bound to Streptavidin Sepharose beads were eluted using biotin and analysed by SDS–PAGE and western blotting against the FLAG-tag in SMG1 and DHX34. Right panel shows a control experiment demonstrating that DHX34 is not eluted when SBP-GFP is used as bait instead of FLAG-SBP-HA-SMG1C. (c) Pull-down experiment testing the interaction of His-UPF1 with FLAG-SBP-HA-SMG1C and FLAG-DHX34. Proteins were mixed and His-UPF1 bound to and eluted from the beads. SDS–PAGE (4–15%) where proteins were identified by western blotting using antibodies against the FLAG and His tags.