protein degradation  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: USP9X promotes the degradation of trapped translation factors on collided ribosomes

doi: 10.64898/2026.04.23.720364

Figure Lengend Snippet: USP9X regulates the occluded A-site RQC pathway (A) Schematic of the CFTR-Y122X-Rluc reporter HEK293 cell line used in genome-wide siRNA and CRISPR screens to identify regulators of NVS1.1 activity. The dual-screen strategy recovered known pathway components (RNF14, RNF25, and GCN1) and the previously uncharacterized factor USP9X. Adapted from Gurzeler et al. (B) Immunoblot analysis and quantification of eRF1 levels in WT and USP9X KO Flp-In 293 HEK cells (KO1 and KO2) treated with 25 μM NVS1.1 or DMSO for 6 hours. Bar graphs show densitometric quantification of eRF1 levels normalized to vinculin (mean ± SD, n = 3). (C) Schematic illustrating the rescue experiment in (D) using the dTAG-USP9X cell line. (D) Immunoblot analysis of dTAG-USP9X cells treated with ± dTAGV-1 (500 nM, 1 hour) and/or NVS1.1 (25 µM, 6 hours). For the rescue experiment, cells were first treated with dTAGV-1 (500 nM, 1 hour), washed with PBS, and incubated in dTAGV-1–free medium for 72 hours (half circle), followed by treatment with DMSO or NVS1.1 at the same concentration and duration as above. (E) Immunoblot analysis and quantification of eEF1A1 levels in WT and USP9X KO cells treated with 50 nM ternatin-4 for 20 hours. Bar graphs show densitometric quantification of eEF1A1 levels normalized to vinculin (mean ± SD, n = 3). (F) Immunoblot analysis of WT, USP9X KO2, and homozygous clones expressing either a silent USP9X variant or a CD USP9X mutant after treatment with 25 μM NVS1.1 for 6 hours.

Article Snippet: Homozygous knock-in clones were subsequently treated with 500 nM dTAGV-1 (MedChemExpress, Cat# HY-145514D) to validate efficient USP9X protein degradation.

Techniques: Genome Wide, CRISPR, Activity Assay, Western Blot, Incubation, Concentration Assay, Clone Assay, Expressing, Variant Assay, Mutagenesis

Journal: bioRxiv

Article Title: USP9X promotes the degradation of trapped translation factors on collided ribosomes

doi: 10.64898/2026.04.23.720364

Figure Lengend Snippet: USP9X depletion partially stabilizes eRF1 following NVS1.1 treatment (A) Immunoblot analysis of Flp-In™ HEK293 cells transfected with two independent USP9X-targeting siRNAs or a non-targeting control siRNA. (B) Schematic representation of the USP9X locus in WT and KI cells. Correctly targeted clones were validated by PCR, yielding products of 443 bp for the WT allele and 1665 bp for the KI allele. PCR products were resolved on a 1% agarose gel. (C) Immunoblot analysis of the positive KI clone following treatment with 500 nM dTAGV-1. (D) Sanger sequencing results of genomic DNA from CRISPR-Cas9 knock-in experiments generating USP9X silent C1566 and catalytic-dead USP9X C1566S cell lines. Sequences were aligned to the WT control sequence.

Article Snippet: Homozygous knock-in clones were subsequently treated with 500 nM dTAGV-1 (MedChemExpress, Cat# HY-145514D) to validate efficient USP9X protein degradation.

Techniques: Western Blot, Transfection, Control, Clone Assay, Agarose Gel Electrophoresis, Sequencing, CRISPR, Knock-In

Journal: bioRxiv

Article Title: USP9X promotes the degradation of trapped translation factors on collided ribosomes

doi: 10.64898/2026.04.23.720364

Figure Lengend Snippet: USP9X KO does not inhibit eRF1 ubiquitination and NVS1.1-mediated pathway activation (A) Immunoblot analysis and quantification of eRF1, eS31, and ubiquitinated eS31 in WT, USP16 KO, and USP9X/USP16 dKO cells treated with 25 μM NVS1.1 for 1 or 6 hours. DMSO-treated cells served as controls. Bar graphs show densitometric quantification of eRF1 levels (normalized to loading control) and the ratio of ubiquitinated to total eS31 (mean ± SD, n = 3). (B) Immunoblot analysis of eRF1 ubiquitination in WT and USP9X KO cells treated with 25 μM NVS1.1 or DMSO for 1 hour. eRF1 was immunoprecipitated, and 50% of the eluate and 1% of the input were analyzed using an anti-eRF1 antibody. (C) Time course of eRF1 ubiquitination in WT and USP9X KO cells treated with 25 μM NVS1.1 for 1 or 6 hours.

Article Snippet: Homozygous knock-in clones were subsequently treated with 500 nM dTAGV-1 (MedChemExpress, Cat# HY-145514D) to validate efficient USP9X protein degradation.

Techniques: Ubiquitin Proteomics, Activation Assay, Western Blot, Control, Immunoprecipitation

Journal: bioRxiv

Article Title: USP9X promotes the degradation of trapped translation factors on collided ribosomes

doi: 10.64898/2026.04.23.720364

Figure Lengend Snippet: Occluded A-site RQC and proteasome remain active in the absence of USP9X (A) Time course analysis of eRF1 ubiquitination in WT and USP9X KO cells treated with 25 μM NVS1.1 for 1, 6, 12, or 24 hours. (B) Fluorometric assay measuring 20S proteasome activity in WT and USP9X KO cells. Activity was assessed using the fluorogenic substrate LLVY-AMC, which releases the fluorescent product 7-amino-4-methylcoumarin (AMC) upon cleavage. AMC fluorescence was measured at an excitation wavelength of 380 nm and an emission wavelength of 460 nm. Lysates treated with the proteasome inhibitor EGCG served as a negative control.

Article Snippet: Homozygous knock-in clones were subsequently treated with 500 nM dTAGV-1 (MedChemExpress, Cat# HY-145514D) to validate efficient USP9X protein degradation.

Techniques: Ubiquitin Proteomics, Activity Assay, Fluorescence, Negative Control

Journal: bioRxiv

Article Title: USP9X promotes the degradation of trapped translation factors on collided ribosomes

doi: 10.64898/2026.04.23.720364

Figure Lengend Snippet: USP9X, RNF14, and RNF25 are required for translational shutdown upon NVS1.1 treatment (A) Puromycin incorporation assay to assess global protein synthesis in WT and USP9X KO cells (two independent homozygous clones). Cells were treated with 25 μM NVS1.1 or DMSO for 6 hours, followed by puromycin (100 μg/mL) for 15 minutes. Cycloheximide (100 μg/mL, 15 minutes) was included as a translation inhibition control. Newly synthesized proteins were detected by immunoblotting with an anti-puromycin antibody; vinculin served as a loading control. (B) Puromycin incorporation assay in WT, USP9X KO2, USP9X silent, and USP9X CD cell lines, performed as in (A). (C) Puromycin incorporation assay in WT, RNF14 KO, and RNF25 KO cell lines, performed as in (A).

Article Snippet: Homozygous knock-in clones were subsequently treated with 500 nM dTAGV-1 (MedChemExpress, Cat# HY-145514D) to validate efficient USP9X protein degradation.

Techniques: Clone Assay, Inhibition, Control, Synthesized, Western Blot

Journal: bioRxiv

Article Title: USP9X promotes the degradation of trapped translation factors on collided ribosomes

doi: 10.64898/2026.04.23.720364

Figure Lengend Snippet: USP9X depletion attenuates NVS1.1-induced translational repression (A) Time course of translation inhibition in WT cells treated with 25 μM NVS1.1, measured by puromycin incorporation. CHX-treated cells were included as a translation inhibition control. (B) Puromycin incorporation assay in WT and USP9X KD cells, performed as in . (C) Quantification of L-azidohomoalanine (L-AHA) incorporation in WT, USP9X silent, and USP9X CD cells treated with 25 μM NVS1.1 or DMSO for 6 hours. L-AHA (100 μM) was added 4 hours after treatment and remained present for the final 2 hours. Bar lines indicate the gating strategy and corresponding percentages of cells without L-AHA incorporation (left) and with incorporated L-AHA (right).

Article Snippet: Homozygous knock-in clones were subsequently treated with 500 nM dTAGV-1 (MedChemExpress, Cat# HY-145514D) to validate efficient USP9X protein degradation.

Techniques: Inhibition, Control

Journal: bioRxiv

Article Title: USP9X promotes the degradation of trapped translation factors on collided ribosomes

doi: 10.64898/2026.04.23.720364

Figure Lengend Snippet: USP9X loss reduces ZNF598 levels and increases poly(A)-induced readthrough (A) Immunoblot analysis and quantification of ZNF598 and eIF4A1 protein levels in WT and USP9X KO cells. Bar graphs show densitometric quantification of ZNF598 and eIF4A1 levels normalized to vinculin (mean ± SD, n = 3). (B) Immunoblot analysis and quantification of ZNF598 protein levels in WT, USP9X silent, and USP9X CD cell lines. Bar graphs show densitometric quantification of ZNF598 abundance normalized to GAPDH (mean ± SD, n = 3). (C) Schematic of dual-fluorescence reporter constructs. The K(20) reporter contains an internal poly(A) tract that induces ribosome stalling, whereas the K(0) reporter lacks the poly(A) sequence and serves as a control.70 (D) Quantification of median RFP:GFP fluorescence ratios in cells expressing stalling or control reporters. Left: comparison of WT, ZNF598 KO, and USP9X KO cells. Right: comparison of WT, ZNF598 KO, and ZNF598/USP9X dKO cells. Each data point represents the median ratio from ∼10,000 cells; error bars show mean ± SD (n = 3).

Article Snippet: Homozygous knock-in clones were subsequently treated with 500 nM dTAGV-1 (MedChemExpress, Cat# HY-145514D) to validate efficient USP9X protein degradation.

Techniques: Western Blot, Fluorescence, Construct, Sequencing, Control, Expressing, Comparison

Journal: bioRxiv

Article Title: USP9X promotes the degradation of trapped translation factors on collided ribosomes

doi: 10.64898/2026.04.23.720364

Figure Lengend Snippet: Catalytically inactive USP9X impairs K6-linked ubiquitination required for VCP recognition (A) DiGly proteomics analysis of cells expressing USP9X-silent or USP9X CD treated with 25 μM NVS1.1 for 6 hours (n = 3). Plotted from Supplemental Table S1. (B) Polysome profiling and proteomic analysis of collided ribosomes in cells treated with or without NVS1.1. Cells were treated with 25 μM NVS1.1 or DMSO for 1 hour, lysed, and subjected to micrococcal nuclease S7 digestion prior to separation through 15%–50% sucrose gradients. Gradients were monitored by A260, and fractions corresponding to disomes and heavy polysomes were collected for mass spectrometry. (C) Differential expression analysis (DEA) identifying proteins enriched in collided ribosome fractions upon NVS1.1 treatment (from B). Plotted from Supplemental Table S2. (D) Ubiquitination analysis of proteins derived from heavy polysome fractions (from B). Plotted from Supplemental Table S3. (E) Immunoblot analysis of HEK293 cells treated with 25 μM NVS1.1 alone or in combination with 10 μM VCP inhibitor for 6 hours.

Article Snippet: Homozygous knock-in clones were subsequently treated with 500 nM dTAGV-1 (MedChemExpress, Cat# HY-145514D) to validate efficient USP9X protein degradation.

Techniques: Ubiquitin Proteomics, Expressing, Mass Spectrometry, Quantitative Proteomics, Derivative Assay, Western Blot

Journal: bioRxiv

Article Title: USP9X promotes the degradation of trapped translation factors on collided ribosomes

doi: 10.64898/2026.04.23.720364

Figure Lengend Snippet: Translation inhibition downstream of the occluded A-Site RQC requires 4EHP (A) Puromycin incorporation assay in WT, 4EHP KO, and 4EHP KO cells with USP9X KD, performed as in . (B) Schematic model of translational repression downstream of ribosome collision. (C) Puromycin incorporation assay in WT, ZNF598 KO, EDF1 KD, GIGYF2 KD, and GIGYF2 KO cells. (D) Polysome profiling of Flp-In 293 HEK cells treated with 25 μM NVS1.1 or DMSO for 1 hour. Lysates were separated on 15%–50% sucrose gradients, and absorbance was recorded at A260. Below, immunoblot analysis of proteins from odd-numbered fractions. Fractions 3 and 5 were diluted 1:15 and 1:3, respectively, prior to analysis. (E) Immunoblot analysis of WT cells treated with anisomycin for 15 minutes (1 mg/L) or 30 minutes (0.5 mg/L), and with 25 μM NVS1.1 for 1 or 6 hours. (F) Puromycin incorporation assay in WT cells treated with 25 μM NVS1.1, 1 μM GCN2 inhibitor, or the combination for 6 hours.

Article Snippet: Homozygous knock-in clones were subsequently treated with 500 nM dTAGV-1 (MedChemExpress, Cat# HY-145514D) to validate efficient USP9X protein degradation.

Techniques: Inhibition, Western Blot

Journal: bioRxiv

Article Title: USP9X promotes the degradation of trapped translation factors on collided ribosomes

doi: 10.64898/2026.04.23.720364

Figure Lengend Snippet: Proposed model of USP9X activity in the occluded A-site RQC Activation of the occluded A-site RQC pathway by NVS1.1 or Ternatin-4 promotes recruitment of RNF25, RNF14, and GCN1 to collided ribosomes bearing an A-site occluded by eRF1 or eEF1A1. RNF14 catalyzes the formation of multiple ubiquitin linkages, including K6-linked chains, which serve as signals for VCP recruitment. VCP subsequently extracts ubiquitinated A-site–trapped factors and facilitates their degradation via the proteasome. Clearance of eRF1 or eEF1A1 triggers a feedback response that inhibits translation initiation through 4EHP, thereby preventing additional rounds of ribosome collisions. In parallel, collided ribosomes are resolved through RNF10-dependent pathways that target them for degradation, as well as ZNF598-mediated rescue mechanisms. Loss of USP9X does not impair ubiquitination per se but disrupts the formation of K6-linked ubiquitin chains. As a consequence, VCP recruitment is compromised, leading to inefficient extraction and degradation. Failure to efficiently clear A-site–trapped factors prevents activation of translational shutdown. In addition, USP9X contributes to the resolution of collided ribosomes by stabilizing ZNF598.

Article Snippet: Homozygous knock-in clones were subsequently treated with 500 nM dTAGV-1 (MedChemExpress, Cat# HY-145514D) to validate efficient USP9X protein degradation.

Techniques: Activity Assay, Activation Assay, Ubiquitin Proteomics, Extraction