FRG1 depletion increases NMD efficiency.

(A–C) Bar graphs depict relative luciferase activity in the NMD reporter under varying conditions of FRG1 manipulation: knockdown (A), knockout (B), and overexpression (C). The figure uses the following abbreviations for labeling: Control_Sc (Control_Scrambled), Control_EV (Control_Empty Vector), WT (Wild Type), FRG1_KD (FRG1 Knockdown), FRG1_KO (FRG1 Knockout), and FRG1_Ex (FRG1 Overexpression). (D-G) Expression of frg1 in wild-type (WT) and frg1 knockout zebrafish at different time points. Image (D) and (E) show frg1 expression in WT and frg1 knockout embryos, respectively, at 24 hours post-fertilization (hpf). Image (F) and (G) depict frg1 expression in WT and frg1 knockout embryos, respectively, at 48 hpf. (H) The bar graph illustrates relative luciferase activity from NMD reporter in the zebrafish embryos having different frg1 expression conditions [wild type (WT), homozygous frg1 knockout (frg1_HOMO_KO), and FRG1 overexpression on frg1 homozygous knockout background (frg1_HOMO_KO+Ex)] at 48 hpf. (I, K) Bar graph depict the proportion of transcripts containing PTCs in MCF7 cell line having FRG1 knockdown (I) and knockout (K). The analysis was conducted using IsoformSwitchAnalyzeR on RNA-Seq data from MCF7 cells following FRG1 depletion. (J, L) The panels show Volcano plots, illustrating the change in isoform fraction (dIF) plotted against the −log10 of the q-value, highlighting differential transcript usage in FRG1-depleted RNA-Seq data from MCF7 cells. Isoforms are classified based on GENCODE (release 47) annotations: those with PTCs are marked in red (TRUE), with regular stop codons in blue (FALSE), and isoforms lacking an annotated open reading frame in gray (NA). The lower panels present density plots showing the distribution of filtered isoforms relative to thresholds of |dIF| > 0.1 and q-value < 0.05. Q-values were calculated using IsoformSwitchAnalyzeR’s DEXSeq-based statistical framework. (M) The UpSet plot illustrates the overlap of upregulated and downregulated isoforms containing PTCs identified in the FRG1 knockdown (KD) and knockout (KO) RNA-Seq datasets. Experiments were performed in triplicate. Results are presented as mean ± SD. ns-non-significant; * represents p-value ≤ 0.05; ** represents p-value ≤ 0.01; **** represents p-value ≤ 0.0001.

NMD-sensitive isoform levels upon FRG1 depletion.

(A-B) The figure shows the isoform expression profile plots displaying isoform structures with annotated Ensembl IDs, isoform expression, and isoform fractions for genes ARL8B (A) and IRF9 (B), in FRG1 knock down (FRG1_KD) set. (C-D). The figure shows the isoform expression profile plots illustrating the isoform structures with annotated Ensembl IDs, isoform expression levels, and isoform fractions for the genes OSBPL7 (panel C) and REEP4 (panel D) in the FRG1 knockout (FRG1_KO) dataset. Red boxes highlight the NMD-sensitive isoforms of the respective genes. (The remaining top 8 genes exhibiting isoform switching are shown in Suppl. Fig. 2 and 3 for FRG1 knockdown and knockout, respectively.)

DUX4 modulates NMD efficiency via FRG1.

The bar graph shows relative luciferase signal from NMD reporter plasmid under DUX4 and FRG1 perturbed conditions [DUX4 uninduced (DUX4_UI), DUX4 expression induced (DUX4_I), DUX4 expression induced and FRG1 knockdown (DUX4_I+FRG1_KD)] in MCF7 cells. Experiments were performed in triplicate. Results are presented as mean ± SD. * represents p-value ≤ 0.05; ** represents p-value ≤ 0.01.

FRG1 expression perturbation alters UPF1 expression.

(A-B) qRT-PCR expression data (A) and Western blot images (B) showing UPF1 expression upon FRG1 depletion in MCF7 cells. (C-D) qRT-PCR expression data (C) and Western blot images (D) showing UPF1 levels upon ectopic FRG1 expression in MCF7 cells. Here are the details of abbreviations used in labeling: FRG1 knockdown (FRG1_KD), FRG1 overexpression (FRG1_Ex), Control_Scrambled (Control_Sc)/ Control_Empty Vector (Control_EV). Experiments were performed in triplicate. (E) Bar graph depicts the qRT-PCR based expression levels of upf1 following knockout of frg1 (frg1_ko) in zebrafish. (F) Bar graph shows the qRT-PCR based expression levels of upf1 following ectopic expression of FRG1 under frg1 knockout background (frg1_ko+Ex) in zebrafish. Results are presented as mean ± SD. ns-nonsignificant; * represents p-value ≤ 0.05; *** represents p-value ≤ 0.001; **** represents p-value ≤ 0.0001.

FRG1 Facilitates UPF1 Protein Reduction Independent of DUX4 Activity.

(A) Western blot analysis illustrating UPF1 protein levels in MCF7 cells under various conditions: lane-2 shows the effect of DUX4 expression (DUX4_I), while subsequent lanes depict the impact of FRG1 knockdown in the presence of DUX4 expression (DUX4_I + FRG1_KD). (B) Western blot showing time-dependent effect of MG132 treatment (8, 12, and 24 h) on UPF1 ubiquitination in FRG1-overexpressing MCF7 cells. UPF1 was immunoprecipitated and probed with anti-ubiquitin antibody to assess polyubiquitination levels. (displays representative Western blot results showing UPF1 expression in MCF7 cells with endogenous DUX4 expression (DUX4_UI) and overexpression of FRG1 (FRG1_Ex), following treatment with MG132 (50 µM) (Lane-2). Lane-3 illustrates UPF1 expression in MCF7 cells co-expressing DUX4 and FRG1 (DUX4_I + FRG1_Ex). Experiments were performed in triplicate. Results are presented as mean ± SD. ns-nonsignificant; *** represents p-value ≤ 0.001; **** represents p-value ≤ 0.0001.C)

FRG1 interacts with UPF1 and mediates its proteasomal degradation.

(A-B) Co-immunoprecipitation with UPF1 (A) and FRG1 (B) antibodies and representative Western blots showing interaction of FRG1 with UPF1 in MCF7 cell line. (C) Immunoblot analysis of a quantitative co-immunoprecipitation assay using UPF1 antibody demonstrates UPF1 ubiquitination under conditions of FRG1 knockdown (FRG1_KD, left three lanes) and FRG1 overexpression (FRG1_Ex, right three lanes). Co-immunoprecipitation with IgG was used as negative control. (D-F) Validation of FRG1-dependent regulation of UPF1 ubiquitination and stability via the proteasome pathway. (D) Immunoblot analysis of a quantitative co-immunoprecipitation assay using UPF1 antibody demonstrates UPF1 ubiquitination (UB) under conditions of FRG1 endogenous levels (FRG1_WT, left three lanes) and FRG1 knockout (FRG1_KO, right three lanes). Co-immunoprecipitation with IgG was used as negative control. (E) Immunoblot analysis of a quantitative co-immunoprecipitation assay using UPF1 antibody shows comparison of UPF1 ubiquitination (UB) in FRG1 knockout background (FRG1_KO left three lanes) with reintroduction of FRG1 in the knockout background (FRG1_KO+Oex, right three lanes) . (F) Immunoblot analysis of a quantitative co-immunoprecipitation assay using a UPF1 antibody compares UPF1 ubiquitination (UB) levels in FRG1-overexpressing cells (FRG1_Ex, left three lanes) with those in FRG1-overexpressing cells subjected to an 8-hour treatment with 50 µM MG132 (FRG1_OEX_MG132 TRT).

Interaction of FRG1 with Spliceosome and Exon Junction Complex.

(A-B) Representative Western blots demonstrating co-immunoprecipitation with the PRP8 antibody in the nuclear extracts (NE) of MCF7 cells. Panel (A) shows results from cells with FRG1 overexpression, while panel (B) displays findings from cells with FRG1 knockdown. Co-immunoprecipitation with IgG was used as a negative control. (C-D) Representative Western blots illustrating co-immunoprecipitation with the eIF4A3 antibody in the cytoplasmic extract (CE) of MCF7 cells with ectopic FRG1 expression (C) and reduced FRG1 expression (D). Co-immunoprecipitation with IgG was used as a negative control. (E) Upper panel: Sucrose gradient fractionation of cytoplasmic extracts from MCF7 cells. The polysome profile displays relative absorbance at 254 nm across a 20–40% sucrose gradient, Lower panel: Immunoblot analysis of gradient fractions using antibodies against FRG1 and eIF4A3. Immunoblot lanes corresponding to the 40S, 60S, 80S ribosomal subunits, and polysome peaks are indicated. (F) Proximity Ligation Assay (PLA) performed using antibodies against FRG1 and eIF4A3. Representative confocal images of MCF7 cells (acquired at 60× magnification) are shown in the upper panel. Red puncta indicate positive PLA signals, representing protein–protein proximity (<40 nm), suggestive of a potential interaction. Nuclei are counterstained with DAPI (blue). The lower panel shows 100× magnified views of the PLA puncta, emphasizing the specificity of the observed interaction.