Fig 1: OTUD4 interacts with RNA. (A) Poly(A)-RNA was purified with oligo(dT)-beads from HEK293T cells and co-purified OTUD4 protein was visualized by anti-OTUD4 western blotting (WB). RNase treatment (50 µg/ml) reduced the amount of OTUD4 collected on oligo(dT)-beads, while control treatment at 37°C did not. A representative result of four independent experiments is shown. (B) Recombinant GST–OTUD4 binds RNA. GST–OTUD4 protein or GST alone were coupled to glutathione–Sepharose and incubated with biotinylated RNA or without RNA. Binding was detected by incubation with fluorescently labeled streptavidin. The mean±s.e.m. of three independent experiments is shown. (C) RNA-immunoprecipitation (RNA-IP) leads to enrichment of RNA with OTUD4. Anti-FLAG IP was performed from lysates of control (vector)- or FLAG–OTUD4-transfected cells. The amount of isolated RNA with OTUD4 is shown in relation to RNA purified in the control samples. Shown is the mean±s.e.m. from n=6 experiments. (D) Detection of OTUD4 mRNA bound to OTUD4. RNA-IP was performed from FLAG–OTUD4-transfected HEK293T cells under stringent conditions. RNA was extracted from IgG-control beads or anti-FLAG beads, and equal RNA volumes were used for RT-PCR reactions. To avoid overamplification of transfected OTUD4 RNA, primers for the 3'UTR of OTUD4-mRNA were chosen, which is not part of the overexpression construct. RT-PCR was performed from two independent RNA-IPs. (E) Domain structure of OTUD4. Top panel: OTUD4 contains an ovarian tumor (OTU) domain that provides activity to cleave ubiquitin chains as well as a putative Tudor-like domain. PONDR-VSL2 (middle panel) and foldIndex (bottom panel) algorithms predict that large portions of OTUD4 are disordered or unfolded.
Fig 2: The clinical relevance of the OTUD4/GSDME axis and radiotherapy response in NPC. A Representative immunohistochemical images of OTUD4 low- and high- expression in NPC tissues (left and right, respectively). The magnified inset area is shown at the bottom. Scale bars represent 50 µm. B The IHC score of OTUD4 in the radiosensitive and radioresistant tissues. C The association between OTUD4 expression and radiotherapy response, ?2 test. RS, radiosensitive; RR, radioresistant. D 5-year PFS for NPC was calculated by Kaplan–Meier analysis, compared using the Log Rank test, and stratified by low and high OTUD4 levels. Progression-free survival, PFS. E Representative NPC cases received radiotherapy alone, showing the relationship between OTUD4 expression, GSDME expression, serum LDH, and cancer regression. The yellow dashed lines indicate tumor in nasopharyngoscopy images. Scale bars represent 50 µm. RT, radiotherapy. F The correlation between OTUD4 expression and GSDME expression. G The correlation between OTUD4/GSDME expression and radiosensitivity. H Comparison of the progression-free survival and locoregional recurrence-free survival between 39 OTUD4low/GSDMElow, 56 OTUD4high/GSDME.high and 55 others. Actuarial probabilities were analyzed by Kaplan–Meier (Log Rank test). (I) Proposed model showing that GSDME-dependent pyroptosis is identified as a critical determinant of radiosensitivity in NPC. In addition, OTUD4/GSDME interaction inhibits OTUD4-mediated GSDME ubiquitination and stability, thereby promoting GSDME-dependent pyroptosis and enhancing radiosensitivity in NPC. *P < 0.05, **P < 0.01, ****P < 0.0001
Fig 3: OTUD4 interactome analysis places OTUD4 in a network of stress granule-associated proteins and RBPs. (A) Identification of putative OTUD4-interacting proteins from mouse cerebellum and cortex lysate by mass spectrometry. Purified HA–OTUD4 or a HA-tagged control peptide were used as bait to pulldown interacting proteins from mouse brain lysates. Potential interactors were identified by mass spectrometry. An enrichment index for hits from at least two out of three experiments (per tissue) was calculated vs controls. Proteins with more than 30-fold enrichment over controls were considered as potential OTUD4 interactors. 290 proteins were identified in the cortex samples and 298 in the cerebellum samples with 133 proteins found in both tissues (Tables S1 and S2). 40% of these have been found in stress granules in previous studies (Jain et al., 2016; Markmiller et al., 2018) and 68% in mRNA interactomes (Beckmann et al., 2015). (B) Gene ontology analysis with the PANTHER GO slim tool (molecular function) revealed a strong enrichment of RNA-binding-related terms for the OTUD4-interacting proteins. Shown is the fold enrichment of terms compared to the mouse reference genome; depicted are all terms with an enrichment factor >5. The false discovery rate (FDR) for each of the terms is indicated in the respective bar. The analysis was performed for 298 potential interactors from cerebellum. (C–G) Co-IP experiments to check interactions of putative binding partners with OTUD4. (C) Endogenous OTUD4 was precipitated from HeLa cell lysates. Lysates were incubated at 37°C for 15 min in the absence or presence of 50 µg/ml RNase A. Co-precipitation of endogenous SMN1 was detected by western blotting. (D–G) HEK293T cells were transfected as indicated and lysates treated as above. Tagged OTUD4 was precipitated. Co-purification of exogenously expressed HuB (D) and IGF2BP3 (E) was confirmed, as well as of cellular G3BP1 (F) and TIAR (G). Treatment with RNase A (third lane of each panel) reduced or abolished the interaction with SMN1, HuB and G3BP1, demonstrating that these interactions are RNA dependent. In contrast, TIAR and IGF2BP3 seem to bind in an RNA-independent manner (* denotes cross-reaction of anti-TIAR-antibody with GFP). A representative of at least three independent experiments is shown for each IP.
Fig 4: OTUD4 is part of mobile neuronal RNA granules. (A) Primary rat hippocampal neurons were transfected with EGFP–OTUD4 (green) and stained with anti-FMRP antibody (red) at days in vitro 4 (DIV4). Shown is an overlay of both channels, while the individual channels are shown for the enlargement of the boxed region (straightened). Magnification of the overlay image illustrates colocalization with FMRP. The EGFP control does not show neuronal granules (Fig. S3A). The experiment was performed five times. For quantification of colocalization, a total of 3204 OTUD4-containing granules were counted in ten neurons from two independent experiments. After filtering, 2433 OTUD4-positive granules were quantified as positive for a FMRP signal (76±11.8%, mean±s.d.). Scale bar: 10 µm. (B) OTUD4-positive granules contain RNA. Shown is a representative rat hippocampal neuron, transfected with FLAG–OTUD4. In situ hybridization with Cy3-oligo(dT) probe visualizes mRNA, and cells are co-stained with anti-FLAG antibody for FLAG-OTUD4 (DIV4). Magnifications of boxed regions (straightened) are shown below the overview picture. Note: to be able to visualize granules in the neurites the signal in the cell body had to be overexposed. The experiment was performed two times. A control Cy3-oligo(dT) hybridization of a neuron transfected with empty FLAG vector is shown in Fig. S3C. Scale bar: 20 µm. (C) mOrange2–OTUD4 does not colocalize with stress granule marker TIAR. Rat hippocampal neuron (DIV4), transfected with mOrange2–OTUD4 (red) and stained for TIAR (green). The image is an overlay of both channels. Scale bar: 5 µm. (D) Live-cell imaging of rat hippocampal neuron (DIV4), transfected with mOrange2–OTUD4. The ROI was straightened for illustration. Tracing shows anterograde movement of an OTUD4 granule (arrowhead), followed for 37 s. Scale bar: 20 µm. At least 30 neurons from three individual experiments were recorded. See also Movie 1. (E) Kymograph for the neurite presented in D. Vertical lines represent static granules, transversal lines demonstrate anterograde or retrograde movements of OTUD4 granules, recorded over a time course of 4 min. Line thickness correlates to granule size. (F) The mobility of 364 OTUD4-containing granules from a total of 13 neurons from three experiments was analyzed. Granule behavior was grouped in three classes, and results were visualized in a pie chart. Quantifications including standard deviation were: stationary, 35±22%; oscillating, 34±11%; mobile, 30±19%. (G) Hippocampal neuron (DIV4), transfected with mOrange2–OTUD4 and neonGreen–SMN1 for live-cell imaging. The ROI was straightened for illustration and granule movement was followed over 24 s. Most granules seem to contain both proteins. The white arrowhead denotes how a granule containing mOrange2–OTUD4 and neonGreen–SMN1 separates into two apparent units when moving retrogradely, with the neonGreen–SMN1 signal taking the lead. When the movement halts, mOrange2 and neonGreen signals overlap again (see Movie 2). At least 20 neurons from three independent experiments were recorded.
Fig 5: OTUD4 enhances radiosensitivity in NPC cells by promoting GSDME-dependent pyroptosis. A Western blotting showed the protein levels of OTUD4 and GSDME in HONE1 and 5-8F cells transfected with or without OTUD4 and shGSDME. B-G The above cells were exposed to the indicated irradiation dose, and then (B) phase-contrast cell imaging, (C) LDH release assay, (D) live cell imaging, (E) Annexin/PI assay, and (F) colony formation assay was performed at designated time points. G The dose-survival curves of the above cells are indicated. All data are presented as the mean ± SD of three independent experiments. H Representative bioluminescence images of tumors were taken on day 42. I Statistical analysis of photon flux (n = 5). J Tumor size was monitored every 4 days (n = 5). K Images of resected tumors. L Tumor weight on day 42 (n = 5). M Serum LDH concentrations were determined pre-radiotherapy and following the third and sixth radiotherapy treatments (n = 3). H Representative images of IHC staining of the tumor sections are shown. Scale bars represent 50 µm. ns, No significant difference. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
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