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( A ) Knockdown of MARCHF7 or <t>UBR5</t> resulted in nsp16 restoration. HEK293T cells were transfected with small interfering RNA (siRNA) of E3 ligase candidates for 24 hr, followed by co-incubation with the nsp16-Flag-expressing plasmids for 48 hr, treated with MG132 (10 µM) for 16 hr before harvesting, lysed, and subjected to immunoblotting (IB) assay using anti-Flag antibody. RT-qPCR was conducted to determine the mRNA expression levels of E3 ligase candidates. The siRNA targeting regions for the candidate E3 ubiquitin ligase proteins and the targeted regions for RT-qPCR are shown in . Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( B ) RNA levels of UBR5 or MARCHF7 from HEK293T cells infected with lentivirus containing control or shRNA targeting UBR5 or MARCHF7 for 48 hr and screened with antibiotics for 48 hr. Knockdown cell lines were transfected with plasmids expressing nsp16-Flag, collected at the indicated times, and the protein levels of nsp16, MARCHF7, and UBR5 were detected by IB. ( C ) MARCHF7 and UBR5 acted separately and did not depend on each other. HEK293T cells stably expressing UBR5 shRNA or MARCHF7 shRNA were transfected with siRNA of MARCHF7 or UBR5 for 24 hr, respectively, followed by co-incubation with the nsp16-Flag-expressing plasmids for 48 hr. The protein levels and the RNA levels of nsp16, UBR5, and MARCHF7 were measured by IB and RT-qPCR, respectively. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( D, E ) In HEK293T cells stably expressing UBR5 shRNA or MARCHF7 shRNA, nsp16 was degraded by overexpressed UBR5 or MARCHF7, respectively, whereas the mutant failed to degrade nsp16. The cell lysates were analyzed by anti-Flag antibody. Figure 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—source data 2. Original files for western blot analysis displayed in . Figure 2—source data 3. Numerical data obtained during experiments represented in .

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1) Product Images from "SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7"

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

Journal: eLife

doi: 10.7554/eLife.102277

( A ) Knockdown of MARCHF7 or UBR5 resulted in nsp16 restoration. HEK293T cells were transfected with small interfering RNA (siRNA) of E3 ligase candidates for 24 hr, followed by co-incubation with the nsp16-Flag-expressing plasmids for 48 hr, treated with MG132 (10 µM) for 16 hr before harvesting, lysed, and subjected to immunoblotting (IB) assay using anti-Flag antibody. RT-qPCR was conducted to determine the mRNA expression levels of E3 ligase candidates. The siRNA targeting regions for the candidate E3 ubiquitin ligase proteins and the targeted regions for RT-qPCR are shown in . Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( B ) RNA levels of UBR5 or MARCHF7 from HEK293T cells infected with lentivirus containing control or shRNA targeting UBR5 or MARCHF7 for 48 hr and screened with antibiotics for 48 hr. Knockdown cell lines were transfected with plasmids expressing nsp16-Flag, collected at the indicated times, and the protein levels of nsp16, MARCHF7, and UBR5 were detected by IB. ( C ) MARCHF7 and UBR5 acted separately and did not depend on each other. HEK293T cells stably expressing UBR5 shRNA or MARCHF7 shRNA were transfected with siRNA of MARCHF7 or UBR5 for 24 hr, respectively, followed by co-incubation with the nsp16-Flag-expressing plasmids for 48 hr. The protein levels and the RNA levels of nsp16, UBR5, and MARCHF7 were measured by IB and RT-qPCR, respectively. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( D, E ) In HEK293T cells stably expressing UBR5 shRNA or MARCHF7 shRNA, nsp16 was degraded by overexpressed UBR5 or MARCHF7, respectively, whereas the mutant failed to degrade nsp16. The cell lysates were analyzed by anti-Flag antibody. Figure 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—source data 2. Original files for western blot analysis displayed in . Figure 2—source data 3. Numerical data obtained during experiments represented in .

Figure Legend Snippet: ( A ) Knockdown of MARCHF7 or UBR5 resulted in nsp16 restoration. HEK293T cells were transfected with small interfering RNA (siRNA) of E3 ligase candidates for 24 hr, followed by co-incubation with the nsp16-Flag-expressing plasmids for 48 hr, treated with MG132 (10 µM) for 16 hr before harvesting, lysed, and subjected to immunoblotting (IB) assay using anti-Flag antibody. RT-qPCR was conducted to determine the mRNA expression levels of E3 ligase candidates. The siRNA targeting regions for the candidate E3 ubiquitin ligase proteins and the targeted regions for RT-qPCR are shown in . Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( B ) RNA levels of UBR5 or MARCHF7 from HEK293T cells infected with lentivirus containing control or shRNA targeting UBR5 or MARCHF7 for 48 hr and screened with antibiotics for 48 hr. Knockdown cell lines were transfected with plasmids expressing nsp16-Flag, collected at the indicated times, and the protein levels of nsp16, MARCHF7, and UBR5 were detected by IB. ( C ) MARCHF7 and UBR5 acted separately and did not depend on each other. HEK293T cells stably expressing UBR5 shRNA or MARCHF7 shRNA were transfected with siRNA of MARCHF7 or UBR5 for 24 hr, respectively, followed by co-incubation with the nsp16-Flag-expressing plasmids for 48 hr. The protein levels and the RNA levels of nsp16, UBR5, and MARCHF7 were measured by IB and RT-qPCR, respectively. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( D, E ) In HEK293T cells stably expressing UBR5 shRNA or MARCHF7 shRNA, nsp16 was degraded by overexpressed UBR5 or MARCHF7, respectively, whereas the mutant failed to degrade nsp16. The cell lysates were analyzed by anti-Flag antibody. Figure 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—source data 2. Original files for western blot analysis displayed in . Figure 2—source data 3. Numerical data obtained during experiments represented in .

Techniques Used: Knockdown, Transfection, Small Interfering RNA, Incubation, Expressing, Western Blot, Quantitative RT-PCR, Ubiquitin Proteomics, Two Tailed Test, Infection, Control, shRNA, Stable Transfection, Mutagenesis

( A, B ) UBR5 small interfering RNA (siRNA) was transfected into shMARCHF7 cells to knock down UBR5 . After 24 hr, MARCHF7 and nsp16 expression vectors were co-transfected, and the cells were harvested 72 hr later. The levels of nsp16 were characterized by immunoblotting (IB) with anti-Flag antibody. Whether MARCHF7 was dependent on UBR5 to degrade nsp16 was determined by further transfection of MARCHF7 siRNA into shUBR5 cells, followed by co-transfection of UBR5 and nsp16 expression vectors 24 hr later. The other operations are the same as above. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test. p>0.05; **p<0.01; ***p<0.001. ( C ) The siRNA targeting regions and RT-qPCR targeting regions for the E3 ubiquitin ligases—HECTD1, UBR5, MYCBP2, TRIM21, TRIM32, and MARCHF7—are shown. Figure 2—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 2—figure supplement 1—source data 3. Numerical data obtained during experiments represented in .

Figure Legend Snippet: ( A, B ) UBR5 small interfering RNA (siRNA) was transfected into shMARCHF7 cells to knock down UBR5 . After 24 hr, MARCHF7 and nsp16 expression vectors were co-transfected, and the cells were harvested 72 hr later. The levels of nsp16 were characterized by immunoblotting (IB) with anti-Flag antibody. Whether MARCHF7 was dependent on UBR5 to degrade nsp16 was determined by further transfection of MARCHF7 siRNA into shUBR5 cells, followed by co-transfection of UBR5 and nsp16 expression vectors 24 hr later. The other operations are the same as above. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test. p>0.05; **p<0.01; ***p<0.001. ( C ) The siRNA targeting regions and RT-qPCR targeting regions for the E3 ubiquitin ligases—HECTD1, UBR5, MYCBP2, TRIM21, TRIM32, and MARCHF7—are shown. Figure 2—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 2—figure supplement 1—source data 3. Numerical data obtained during experiments represented in .

Techniques Used: Small Interfering RNA, Transfection, Knockdown, Expressing, Western Blot, Cotransfection, Two Tailed Test, Quantitative RT-PCR, Ubiquitin Proteomics

( A ) Nsp16 can be ubiquitinated. HEK293T cells co-transfected with ubiquitin-Myc and nsp16-Flag or transfected with nsp16-Flag alone. The cells were treated with MG132 for 12 hr before collection. The whole-cell lysates were incubated with anti-Flag beads and used for immunoblotting (IB) with anti-Myc or anti-Flag antibodies to detect the polyubiquitination chain of nsp16. ( B ) Assess the endogenous ubiquitination level of nsp16 protein. Cells were transfected with nsp16-Flag or an empty vector and collected 48 hr later. Prior to harvesting, cells were treated with MG132 for 16 hr. Co-immunoprecipitation (Co-IP) experiments were then performed to analyze the endogenous ubiquitination level of nsp16. ( C ) The level of ubiquitination of nsp16 decreased with decreasing the protein levels of MARCHF7 or UBR5. E3 was knocked down by transfection with small interfering RNA (siRNA) targeting UBR5 or MARCHF7 , and 24 hr later, ubiquitin-Myc and nsp16-HA were co-transfected or nsp16-HA alone. Cells were treated with MG132 for 16 hr before collection. Whole-cell lysates were incubated with anti-HA beads, and polyubiquitinated chains of nsp16 were detected by IB with anti-Myc or anti-HA antibodies. ( D ) Nsp16 can be modified by a variety of ubiquitin chains. HEK293T cells were transfected with either nsp16-HA alone or together with plasmids encoding various mutants of ubiquitin (K6 only, K11 only, K27 only, K29 only, K33 only, K48 only, K63 only). Thirty-six hours later, cells were treated with MG132 for 12 hr. Cell lysates were then subjected to immunoprecipitation, followed by IB to analysis. ( E, F ) MARCHF7 or UBR5 causes nsp16 to be modified by the K27-type or K48-type ubiquitin chain. 293T cell lines with or without MARCHF7 or UBR5 knockdown were co-transfected with plasmids encoding ubiquitin-WT or various mutants of ubiquitin (K6 only, K11 only, K27 only, K29 only, K33 only, K48 only, K63 only). The other experimental methods were the same as C. Figure 3—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—source data 2. Original files for western blot analysis displayed in .

Figure Legend Snippet: ( A ) Nsp16 can be ubiquitinated. HEK293T cells co-transfected with ubiquitin-Myc and nsp16-Flag or transfected with nsp16-Flag alone. The cells were treated with MG132 for 12 hr before collection. The whole-cell lysates were incubated with anti-Flag beads and used for immunoblotting (IB) with anti-Myc or anti-Flag antibodies to detect the polyubiquitination chain of nsp16. ( B ) Assess the endogenous ubiquitination level of nsp16 protein. Cells were transfected with nsp16-Flag or an empty vector and collected 48 hr later. Prior to harvesting, cells were treated with MG132 for 16 hr. Co-immunoprecipitation (Co-IP) experiments were then performed to analyze the endogenous ubiquitination level of nsp16. ( C ) The level of ubiquitination of nsp16 decreased with decreasing the protein levels of MARCHF7 or UBR5. E3 was knocked down by transfection with small interfering RNA (siRNA) targeting UBR5 or MARCHF7 , and 24 hr later, ubiquitin-Myc and nsp16-HA were co-transfected or nsp16-HA alone. Cells were treated with MG132 for 16 hr before collection. Whole-cell lysates were incubated with anti-HA beads, and polyubiquitinated chains of nsp16 were detected by IB with anti-Myc or anti-HA antibodies. ( D ) Nsp16 can be modified by a variety of ubiquitin chains. HEK293T cells were transfected with either nsp16-HA alone or together with plasmids encoding various mutants of ubiquitin (K6 only, K11 only, K27 only, K29 only, K33 only, K48 only, K63 only). Thirty-six hours later, cells were treated with MG132 for 12 hr. Cell lysates were then subjected to immunoprecipitation, followed by IB to analysis. ( E, F ) MARCHF7 or UBR5 causes nsp16 to be modified by the K27-type or K48-type ubiquitin chain. 293T cell lines with or without MARCHF7 or UBR5 knockdown were co-transfected with plasmids encoding ubiquitin-WT or various mutants of ubiquitin (K6 only, K11 only, K27 only, K29 only, K33 only, K48 only, K63 only). The other experimental methods were the same as C. Figure 3—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—source data 2. Original files for western blot analysis displayed in .

Techniques Used: Transfection, Ubiquitin Proteomics, Incubation, Western Blot, Plasmid Preparation, Immunoprecipitation, Co-Immunoprecipitation Assay, Small Interfering RNA, Modification, Knockdown

( A ) HEK293T cells were transfected with either nsp16-Flag alone or together with MARCHF7-Myc. Thirty-six hours after transfection, the cells were treated with MG132 (10 µM) for 12 hr. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody. Using immunoblotting (IB) to analyze the precipitates and input. ( B ) HEK293T cells were transfected with nsp16-Flag. Cell lysates were subjected to immunoprecipitation with anti-UBR5 or IgG antibody. ( C, D ) HeLa cells were co-transfected with YFP-nsp16 and CFP-UBR5 or CFP-MARCHF7. A representative image of YFP-nsp16 (yellow) and ECFP-MARCHF7 (cyan) or ECFP-UBR5 (cyan) expressing cells before and after photobleaching the acceptor fluorophore, YFP. The region chosen for photobleaching is marked (white open box). Scale bars, 10 µm. The quantization of fluorescence brightness was analyzed by ImageJ. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test. p>0.05; **p<0.01; ***p<0.001. ( E ) HeLa cells transfected with nsp16-Flag were analyzed by confocal microscopy. The Flag-tagged nsp16 labeled with anti-Flag antibody (red). MARCHF7 or UBR5 were labeled with endogenous antibodies (green). Cell nuclei were stained using DAPI (4′,6-diamidino-2-phenylindole) (blue). Representative images were shown. Scale bars, 20 µm. The ratio of colocalization was quantified by measuring the fluorescence intensities using ImageJ. ( F ) nsp16 was stably transfected into HEK293T cells. The cells were analyzed by confocal microscopy. The other operations are the same as above. Figure 4—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 4—figure supplement 1—source data 3. Numerical data obtained during experiments represented in .

Figure Legend Snippet: ( A ) HEK293T cells were transfected with either nsp16-Flag alone or together with MARCHF7-Myc. Thirty-six hours after transfection, the cells were treated with MG132 (10 µM) for 12 hr. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody. Using immunoblotting (IB) to analyze the precipitates and input. ( B ) HEK293T cells were transfected with nsp16-Flag. Cell lysates were subjected to immunoprecipitation with anti-UBR5 or IgG antibody. ( C, D ) HeLa cells were co-transfected with YFP-nsp16 and CFP-UBR5 or CFP-MARCHF7. A representative image of YFP-nsp16 (yellow) and ECFP-MARCHF7 (cyan) or ECFP-UBR5 (cyan) expressing cells before and after photobleaching the acceptor fluorophore, YFP. The region chosen for photobleaching is marked (white open box). Scale bars, 10 µm. The quantization of fluorescence brightness was analyzed by ImageJ. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test. p>0.05; **p<0.01; ***p<0.001. ( E ) HeLa cells transfected with nsp16-Flag were analyzed by confocal microscopy. The Flag-tagged nsp16 labeled with anti-Flag antibody (red). MARCHF7 or UBR5 were labeled with endogenous antibodies (green). Cell nuclei were stained using DAPI (4′,6-diamidino-2-phenylindole) (blue). Representative images were shown. Scale bars, 20 µm. The ratio of colocalization was quantified by measuring the fluorescence intensities using ImageJ. ( F ) nsp16 was stably transfected into HEK293T cells. The cells were analyzed by confocal microscopy. The other operations are the same as above. Figure 4—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 4—figure supplement 1—source data 3. Numerical data obtained during experiments represented in .

Techniques Used: Transfection, Immunoprecipitation, Western Blot, Expressing, Fluorescence, Two Tailed Test, Confocal Microscopy, Labeling, Staining, Stable Transfection

( A, B ) The binding of MARCHF7 or UBR5 to nsp16 was not mutually dependent. The binding of nsp16 to UBR5 or MARCHF7 was identified by co-immunoprecipitation in HEK293T cells transfected into si MARCHF7 or si UBR5 , respectively. The immunoprecipitates and input were analyzed by immunoblotting (IB). The knockdown efficiency was detected by RT-qPCR and IB. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( C, D ) MARCHF7 or UBR5 colocalized with nsp16 in the endoplasmic reticulum. Hela cells were co-transfected with YFP-nsp16 (yellow) and CFP-UBR5 (cyan) or CFP-MARCHF7 (cyan). The organelles were labeled with antibodies against marker proteins of endoplasmic reticulum, Golgi apparatus, and mitochondria respectively (red). The cells were analyzed by confocal microscopy ( C ). Scale bars, 20 µm. The ratio of colocalization was quantified by measuring the fluorescence intensities using ImageJ ( D ). Figure 4—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—source data 2. Original files for western blot analysis displayed in . Figure 4—source data 3. Numerical data obtained during experiments represented in .

Figure Legend Snippet: ( A, B ) The binding of MARCHF7 or UBR5 to nsp16 was not mutually dependent. The binding of nsp16 to UBR5 or MARCHF7 was identified by co-immunoprecipitation in HEK293T cells transfected into si MARCHF7 or si UBR5 , respectively. The immunoprecipitates and input were analyzed by immunoblotting (IB). The knockdown efficiency was detected by RT-qPCR and IB. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( C, D ) MARCHF7 or UBR5 colocalized with nsp16 in the endoplasmic reticulum. Hela cells were co-transfected with YFP-nsp16 (yellow) and CFP-UBR5 (cyan) or CFP-MARCHF7 (cyan). The organelles were labeled with antibodies against marker proteins of endoplasmic reticulum, Golgi apparatus, and mitochondria respectively (red). The cells were analyzed by confocal microscopy ( C ). Scale bars, 20 µm. The ratio of colocalization was quantified by measuring the fluorescence intensities using ImageJ ( D ). Figure 4—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—source data 2. Original files for western blot analysis displayed in . Figure 4—source data 3. Numerical data obtained during experiments represented in .

Techniques Used: Binding Assay, Immunoprecipitation, Transfection, Western Blot, Knockdown, Quantitative RT-PCR, Two Tailed Test, Labeling, Marker, Confocal Microscopy, Fluorescence

( A ) The schematic represents UBR5 wild-type (WT) or mutants used in the study. ( B ) The homologous to the E6AP carboxyl terminus (HECT) domain of UBR5 is required for nsp16 degradation. After co-transfection with UBR5 WT or mutants and nsp16-HA, cells were harvested 48 hr later, and cell lysates were examined by immunoblotting (IB). ( C ) The HECT domain of UBR5 affects K48-type ubiquitin chain of nsp16. HEK293T cells were transfected with the assigned plasmids. After 36 hr, cells were treated with 10 µM MG132 for 12 hr, harvested, and cell lysates were incubated with protein G agarose beads conjugated with anti-HA antibodies. Cell lysates and precipitated samples were analyzed by IB. ( D ) The schematic represents WT and truncated forms of MARCHF7 used in the study. (E) Only MARCHF7 WT degraded nsp16. ( F ) The N-terminal region of MARCHF7 interacted with nsp16, and only the WT could catalyze the K27-type ubiquitin chain of nsp16. Figure 4—figure supplement 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Figure Legend Snippet: ( A ) The schematic represents UBR5 wild-type (WT) or mutants used in the study. ( B ) The homologous to the E6AP carboxyl terminus (HECT) domain of UBR5 is required for nsp16 degradation. After co-transfection with UBR5 WT or mutants and nsp16-HA, cells were harvested 48 hr later, and cell lysates were examined by immunoblotting (IB). ( C ) The HECT domain of UBR5 affects K48-type ubiquitin chain of nsp16. HEK293T cells were transfected with the assigned plasmids. After 36 hr, cells were treated with 10 µM MG132 for 12 hr, harvested, and cell lysates were incubated with protein G agarose beads conjugated with anti-HA antibodies. Cell lysates and precipitated samples were analyzed by IB. ( D ) The schematic represents WT and truncated forms of MARCHF7 used in the study. (E) Only MARCHF7 WT degraded nsp16. ( F ) The N-terminal region of MARCHF7 interacted with nsp16, and only the WT could catalyze the K27-type ubiquitin chain of nsp16. Figure 4—figure supplement 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Techniques Used: Cotransfection, Western Blot, Ubiquitin Proteomics, Transfection, Incubation

( A, B ) Knocking down MARCHF7 or UBR5 enhances SARS-CoV-2 trVLP infectivity. MARCHF7 or UBR5 was knocked down by small interfering RNA (siRNA) in Caco2 cells with stable expression of SARS-CoV-2 N protein. Twenty-four hours later, cells were infected with SARS-CoV-2 virus-like particles (MOI: 0.1), the medium was changed 2 hr after infection, and the eGFP-positive cells were detected by flow cytometry 48 hr later ( A ). Protein content was determined by RT-qPCR ( B ). Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest: *p<0.05; **p<0.01; ***p<0.001. Figure 5—figure supplement 1—source data 1. Numerical data obtained during experiments represented in .

Figure Legend Snippet: ( A, B ) Knocking down MARCHF7 or UBR5 enhances SARS-CoV-2 trVLP infectivity. MARCHF7 or UBR5 was knocked down by small interfering RNA (siRNA) in Caco2 cells with stable expression of SARS-CoV-2 N protein. Twenty-four hours later, cells were infected with SARS-CoV-2 virus-like particles (MOI: 0.1), the medium was changed 2 hr after infection, and the eGFP-positive cells were detected by flow cytometry 48 hr later ( A ). Protein content was determined by RT-qPCR ( B ). Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest: *p<0.05; **p<0.01; ***p<0.001. Figure 5—figure supplement 1—source data 1. Numerical data obtained during experiments represented in .

Techniques Used: Infection, Small Interfering RNA, Expressing, Virus, Flow Cytometry, Quantitative RT-PCR

( A ) The virus-encoded nsp16 protein interacts with endogenous MARCHF7 and UBR5 and undergoes ubiquitination modification. In 293T-ACE2 cells, with or without IME-BJ01 strain infection (MOI: 0.01), the medium was changed 2 hr post-infection, and cells were harvested 48 hr later, with MG132 treatment added 16 hr before harvesting. nsp16 protein was enriched using protein G beads coupled with the nsp16 antibody, and interactions and ubiquitination were analyzed by immunoblotting (IB) with endogenous antibodies against MARCHF7, UBR5, and ubiquitination. ( B–I ) MARCHF7 and UBR5 were knocked down by small interfering RNA (siRNA) in Caco2 cells. 24 hr after transfection, the cells were infected with IME-BJ01 strain (MOI: 0.01) ( C–E ) or Omicron BA.1 strain (MOI: 0.001) ( F–H ), respectively. 2 hr post-infection, the supernatant was discarded, and the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 3% fetal bovine serum for 48 hr. The mRNA levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M and E genes in the cells ( C, F ) and E genes in supernatant ( D, G ) were detected by RT-qPCR, and the viral titers in supernatant ( E, H ) were measured. The N protein levels of IME-BJ01 or Omicron viruses were detected by IB ( I ). Knockdown efficiencies of MARCHF7 and UBR5 were detected by RT-qPCR or IB ( B, I ). Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest: *p<0.05; **p<0.01; ***p<0.001. Figure 5—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 5—source data 2. Original files for western blot analysis displayed in . Figure 5—source data 3. Numerical data obtained during experiments represented in .

Figure Legend Snippet: ( A ) The virus-encoded nsp16 protein interacts with endogenous MARCHF7 and UBR5 and undergoes ubiquitination modification. In 293T-ACE2 cells, with or without IME-BJ01 strain infection (MOI: 0.01), the medium was changed 2 hr post-infection, and cells were harvested 48 hr later, with MG132 treatment added 16 hr before harvesting. nsp16 protein was enriched using protein G beads coupled with the nsp16 antibody, and interactions and ubiquitination were analyzed by immunoblotting (IB) with endogenous antibodies against MARCHF7, UBR5, and ubiquitination. ( B–I ) MARCHF7 and UBR5 were knocked down by small interfering RNA (siRNA) in Caco2 cells. 24 hr after transfection, the cells were infected with IME-BJ01 strain (MOI: 0.01) ( C–E ) or Omicron BA.1 strain (MOI: 0.001) ( F–H ), respectively. 2 hr post-infection, the supernatant was discarded, and the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 3% fetal bovine serum for 48 hr. The mRNA levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M and E genes in the cells ( C, F ) and E genes in supernatant ( D, G ) were detected by RT-qPCR, and the viral titers in supernatant ( E, H ) were measured. The N protein levels of IME-BJ01 or Omicron viruses were detected by IB ( I ). Knockdown efficiencies of MARCHF7 and UBR5 were detected by RT-qPCR or IB ( B, I ). Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest: *p<0.05; **p<0.01; ***p<0.001. Figure 5—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 5—source data 2. Original files for western blot analysis displayed in . Figure 5—source data 3. Numerical data obtained during experiments represented in .

Techniques Used: Virus, Ubiquitin Proteomics, Modification, Infection, Western Blot, Small Interfering RNA, Transfection, Cell Culture, Quantitative RT-PCR, Knockdown

( A–H ) UBR5 or MARCHF7 was transfected in 293T cells stably overexpressed with ACE2, and the increased doses of nsp16-Flag were transfected simultaneously. After 24 hr, the cells were infected with IME-BJ01 strains. The mRNA levels of M and E genes of the IME-BJ01 strain in the cells ( A, D ) and E gene in supernatant ( B, E ) were detected by RT-qPCR, as well as the detection of viral titers in supernatant ( C, F ). The N protein of the virus and the overexpression efficiency were detected by IB ( G, H ). Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. p>0.05; **p<0.01; ***p<0.001. shows data related to infection with Omicron BA.1. Figure 6—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—source data 2. Original files for western blot analysis displayed in . Figure 6—source data 3. Numerical data obtained during experiments represented in .

Figure Legend Snippet: ( A–H ) UBR5 or MARCHF7 was transfected in 293T cells stably overexpressed with ACE2, and the increased doses of nsp16-Flag were transfected simultaneously. After 24 hr, the cells were infected with IME-BJ01 strains. The mRNA levels of M and E genes of the IME-BJ01 strain in the cells ( A, D ) and E gene in supernatant ( B, E ) were detected by RT-qPCR, as well as the detection of viral titers in supernatant ( C, F ). The N protein of the virus and the overexpression efficiency were detected by IB ( G, H ). Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. p>0.05; **p<0.01; ***p<0.001. shows data related to infection with Omicron BA.1. Figure 6—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—source data 2. Original files for western blot analysis displayed in . Figure 6—source data 3. Numerical data obtained during experiments represented in .

Techniques Used: Transfection, Stable Transfection, Infection, Quantitative RT-PCR, Virus, Over Expression, Western Blot

( A–H ) In 293T-ACE2 cells, the Really Interesting New Gene (RING) domain deletion mutant of MARCHF7 (MARCHF7-aa 1–542) or the homologous to the E6AP carboxyl terminus (HECT) domain inactivated mutant of UBR5 (UBR5-ΔHECT) were transfected, along with a gradient of nsp16-Flag overexpression. The cells were infected with the IME-BJ01 strain (MOI: 0.01), medium was changed 2 hr post-infection, and cells and supernatants were collected 48 hr after infection. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. p>0.05; **p<0.01; ***p<0.001. Figure 6—figure supplement 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 2—source data 2. Original files for western blot analysis displayed in . Figure 6—figure supplement 2—source data 3. Numerical data obtained during experiments represented in .

Figure Legend Snippet: ( A–H ) In 293T-ACE2 cells, the Really Interesting New Gene (RING) domain deletion mutant of MARCHF7 (MARCHF7-aa 1–542) or the homologous to the E6AP carboxyl terminus (HECT) domain inactivated mutant of UBR5 (UBR5-ΔHECT) were transfected, along with a gradient of nsp16-Flag overexpression. The cells were infected with the IME-BJ01 strain (MOI: 0.01), medium was changed 2 hr post-infection, and cells and supernatants were collected 48 hr after infection. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. p>0.05; **p<0.01; ***p<0.001. Figure 6—figure supplement 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 2—source data 2. Original files for western blot analysis displayed in . Figure 6—figure supplement 2—source data 3. Numerical data obtained during experiments represented in .

Techniques Used: Mutagenesis, Transfection, Over Expression, Infection, Western Blot

( A ) This diagram shows the mutation of nsp16 in different virus subtypes. The amino acid sequences of different SARS-CoV-2 strains were obtained from the National Center for Biotechnology Information (NCBI), and the amino acid sequences of nsp16 of different strains were compared by DNAMAN software. ( B ) Nsp16 mutants can still be regulated by MG132. The mutated nsp16 plasmids were transfected into HEK293T cells. After 36 hr of culture, cells were treated with 10 µm MG132 or DMSO, harvested 12 hr later, and cell lysates were examined by immunoblotting (IB). ( C ) MARCHF7 or UBR5 can degrade nsp16 mutants. After transfecting MARCHF7 or UBR5 small interfering RNA (siRNA) and the mutated nsp16 plasmids, the cells were harvested 48 hr later. The cell lysates were detected by IB. Figure 6—figure supplement 3—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 3—source data 2. Original files for western blot analysis displayed in .

Figure Legend Snippet: ( A ) This diagram shows the mutation of nsp16 in different virus subtypes. The amino acid sequences of different SARS-CoV-2 strains were obtained from the National Center for Biotechnology Information (NCBI), and the amino acid sequences of nsp16 of different strains were compared by DNAMAN software. ( B ) Nsp16 mutants can still be regulated by MG132. The mutated nsp16 plasmids were transfected into HEK293T cells. After 36 hr of culture, cells were treated with 10 µm MG132 or DMSO, harvested 12 hr later, and cell lysates were examined by immunoblotting (IB). ( C ) MARCHF7 or UBR5 can degrade nsp16 mutants. After transfecting MARCHF7 or UBR5 small interfering RNA (siRNA) and the mutated nsp16 plasmids, the cells were harvested 48 hr later. The cell lysates were detected by IB. Figure 6—figure supplement 3—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 3—source data 2. Original files for western blot analysis displayed in .

Techniques Used: Mutagenesis, Virus, Software, Transfection, Western Blot, Small Interfering RNA

( A–C ) The protein and mRNA levels of MARCHF7 or UBR5 upon infection with different titers. Endogenous MARCHF7 and UBR5 RNA levels were detected by RT-qPCR 48 hr after infection with different titers of IME-BJ01 strain (MOI: 0, 0.0001, 0.001, 0.01) or Omicron BA.1 strain (MOI: 0, 0.0001, 0.001). Protein levels were examined by immunoblotting (IB). ( D ) The expression level of UBR5 was negatively correlated with the severity of the disease, but MARCHF7 expression levels were not. Peripheral blood mononuclear cells (PBMCs) were extracted from common, severe, and critical coronavirus disease 2019 (COVID-19) patients. RT-qPCR was used to detect the mRNA level of UBR5 or MARCHF7 in patients. Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. ns, p>0.05; **p<0.01; ***p<0.001. Figure 6—figure supplement 4—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 4—source data 2. Original files for western blot analysis displayed in . Figure 6—figure supplement 4—source data 3. Numerical data obtained during experiments represented in .

Figure Legend Snippet: ( A–C ) The protein and mRNA levels of MARCHF7 or UBR5 upon infection with different titers. Endogenous MARCHF7 and UBR5 RNA levels were detected by RT-qPCR 48 hr after infection with different titers of IME-BJ01 strain (MOI: 0, 0.0001, 0.001, 0.01) or Omicron BA.1 strain (MOI: 0, 0.0001, 0.001). Protein levels were examined by immunoblotting (IB). ( D ) The expression level of UBR5 was negatively correlated with the severity of the disease, but MARCHF7 expression levels were not. Peripheral blood mononuclear cells (PBMCs) were extracted from common, severe, and critical coronavirus disease 2019 (COVID-19) patients. RT-qPCR was used to detect the mRNA level of UBR5 or MARCHF7 in patients. Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. ns, p>0.05; **p<0.01; ***p<0.001. Figure 6—figure supplement 4—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 4—source data 2. Original files for western blot analysis displayed in . Figure 6—figure supplement 4—source data 3. Numerical data obtained during experiments represented in .

Techniques Used: Infection, Quantitative RT-PCR, Western Blot, Expressing

( A–G ) BLAB/C mice were injected with the corresponding plasmids at 40 µg/500 µl via the high-pressure tail vein, followed by nasal inoculation with 50 µl severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus at a dosage of 10 5.5 TCID50/ml (created with BioRender.com and the agreement no. is OO281XWHNA). Immunoblotting (IB) was used to detect the expression of MARCHF7 or UBR5 in the lung tissues ( B ). Viral RNA loads in mouse lung tissues were detected by measuring the mRNA levels of the E genes by RT-qPCR ( C ). Lung tissue was collected, homogenized, and the residue was removed by centrifugation to collect the supernatant. The viral titer was then measured using the TCID50 method ( D ). Mouse body weight was monitored during the experimental period ( E ). Representative images of hematoxylin and eosin (H&E) staining of lungs of mice with different treatments. Magnification, ×40. Scale bars, 20 µm ( F ). The staining of viral N proteins. Magnification, ×63. Scale bars, 20 µm. n=3 in each group ( G ). RT-qPCR was used to measure the expression of cytokines and chemokines in the spleens of mice in each group ( H ). Statistical significance was analyzed using a one-way analysis of variance with Tukey’s multiple comparisons test (NS, no significance, *p<0.05, **p<0.01, ***p<0.001). Figure 7—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 7—source data 2. Original files for western blot analysis displayed in . Figure 7—source data 3. Numerical data obtained during experiments represented in .

Figure Legend Snippet: ( A–G ) BLAB/C mice were injected with the corresponding plasmids at 40 µg/500 µl via the high-pressure tail vein, followed by nasal inoculation with 50 µl severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus at a dosage of 10 5.5 TCID50/ml (created with BioRender.com and the agreement no. is OO281XWHNA). Immunoblotting (IB) was used to detect the expression of MARCHF7 or UBR5 in the lung tissues ( B ). Viral RNA loads in mouse lung tissues were detected by measuring the mRNA levels of the E genes by RT-qPCR ( C ). Lung tissue was collected, homogenized, and the residue was removed by centrifugation to collect the supernatant. The viral titer was then measured using the TCID50 method ( D ). Mouse body weight was monitored during the experimental period ( E ). Representative images of hematoxylin and eosin (H&E) staining of lungs of mice with different treatments. Magnification, ×40. Scale bars, 20 µm ( F ). The staining of viral N proteins. Magnification, ×63. Scale bars, 20 µm. n=3 in each group ( G ). RT-qPCR was used to measure the expression of cytokines and chemokines in the spleens of mice in each group ( H ). Statistical significance was analyzed using a one-way analysis of variance with Tukey’s multiple comparisons test (NS, no significance, *p<0.05, **p<0.01, ***p<0.001). Figure 7—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 7—source data 2. Original files for western blot analysis displayed in . Figure 7—source data 3. Numerical data obtained during experiments represented in .

Techniques Used: Injection, Virus, Western Blot, Expressing, Quantitative RT-PCR, Residue, Centrifugation, Staining

Schematic diagram of MARCHF7 and UBR5 ubiquitinate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein nsp16, leading to its degradation via the proteasomal pathway, thereby affecting viral replication (created with BioRender.com and the agreement no. is EV281XWATL).

Figure Legend Snippet: Schematic diagram of MARCHF7 and UBR5 ubiquitinate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein nsp16, leading to its degradation via the proteasomal pathway, thereby affecting viral replication (created with BioRender.com and the agreement no. is EV281XWATL).

Techniques Used:

( A ) Single-lysine mutation of nsp16 protein can be restored by MG132. Single-lysine mutants of nsp16 protein were obtained through mutagenesis and overexpressed in 293T cells. After 36 hr, cells were treated with either MG132 or DMSO for 16 hr and then harvested. Protein levels were detected by immunoblotting (IB) using the HA antibody. ( B ) nsp16 protein truncates can be restored by MG132. nsp16 protein truncates were obtained through structural analysis and mutations. IB was performed to determine whether the truncates could be restored by MG132. ( C ) The mass spectrometry analysis identified the ubiquitination modification site at lysine 76. nsp16-Flag was overexpressed in 293T cells, followed by MG132 treatment and cell harvest. nsp16 protein was enriched using Flag antibody-conjugated protein G beads. Flag-peptide competition was used to obtain the nsp16-containing solution. The protein and ubiquitination status were visualized by SDS-PAGE and Coomassie staining. Mass spectrometry was used for further analysis. ( D ) Degradation of nsp16-K76R is still regulated by MARCHF7 or UBR5. A plasmid with a mutation at lysine 76 of nsp16 to arginine (nsp16-K76R) was constructed. MARCHF7 or UBR5 was knocked down by small interfering RNA (siRNA) in 293T cells. nsp16-WT or nsp16-K76R was transfected the next day, and cells were harvested 48 hr later. Protein levels were detected by IB. ( E ) Ubiquitination levels of nsp16-K76R are reduced but still regulated by MARCHF7 or UBR5. MARCHF7 or UBR5 was knocked down using siRNA in 293T cells. The cells were co-transfected with Ub-K27 or K48, and nsp16-WT or nsp16-K76R mutant. Cells were harvested 48 hr later, with MG132 treatment 16 hr before harvesting. Co-immunoprecipitation (Co-IP) experiments were performed to analyze the ubiquitination status of nsp16-WT or its mutant. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. p>0.05; **p<0.01; ***p<0.001. Figure 8—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 8—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 8—figure supplement 1—source data 3. Numerical data obtained during experiments represented in .

Figure Legend Snippet: ( A ) Single-lysine mutation of nsp16 protein can be restored by MG132. Single-lysine mutants of nsp16 protein were obtained through mutagenesis and overexpressed in 293T cells. After 36 hr, cells were treated with either MG132 or DMSO for 16 hr and then harvested. Protein levels were detected by immunoblotting (IB) using the HA antibody. ( B ) nsp16 protein truncates can be restored by MG132. nsp16 protein truncates were obtained through structural analysis and mutations. IB was performed to determine whether the truncates could be restored by MG132. ( C ) The mass spectrometry analysis identified the ubiquitination modification site at lysine 76. nsp16-Flag was overexpressed in 293T cells, followed by MG132 treatment and cell harvest. nsp16 protein was enriched using Flag antibody-conjugated protein G beads. Flag-peptide competition was used to obtain the nsp16-containing solution. The protein and ubiquitination status were visualized by SDS-PAGE and Coomassie staining. Mass spectrometry was used for further analysis. ( D ) Degradation of nsp16-K76R is still regulated by MARCHF7 or UBR5. A plasmid with a mutation at lysine 76 of nsp16 to arginine (nsp16-K76R) was constructed. MARCHF7 or UBR5 was knocked down by small interfering RNA (siRNA) in 293T cells. nsp16-WT or nsp16-K76R was transfected the next day, and cells were harvested 48 hr later. Protein levels were detected by IB. ( E ) Ubiquitination levels of nsp16-K76R are reduced but still regulated by MARCHF7 or UBR5. MARCHF7 or UBR5 was knocked down using siRNA in 293T cells. The cells were co-transfected with Ub-K27 or K48, and nsp16-WT or nsp16-K76R mutant. Cells were harvested 48 hr later, with MG132 treatment 16 hr before harvesting. Co-immunoprecipitation (Co-IP) experiments were performed to analyze the ubiquitination status of nsp16-WT or its mutant. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. p>0.05; **p<0.01; ***p<0.001. Figure 8—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 8—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 8—figure supplement 1—source data 3. Numerical data obtained during experiments represented in .

Techniques Used: Mutagenesis, Western Blot, Mass Spectrometry, Ubiquitin Proteomics, Modification, SDS Page, Staining, Plasmid Preparation, Construct, Small Interfering RNA, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay

Image Search Results

UBR5 interacted with Snail. (A) UBR5 was positively correlated with Snail in colon adenocarcinoma and rectal adenocarcinoma. Correlation analysis for TCGA and GTEx on the GEPIA website showed a correlation coefficient of R = 0.26, P = 4.4e-13. P < 0.01 denoted statistical significance. (B) The expression levels of UBR5 and Snail correlated with colorectal cancer (CRC) stages. The UBR5 and Snail mRNA levels based on pathological stages were analyzed using the GEPIA2 violin-plots in colorectal tumors. (C) Co-immunoprecipitation assay showed that UBR5 interacted with Snail. HEK293T cells were transfected with Myc-tagged UBR5 and Flag-tagged Snail and treated with MG132 as indicated. Cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibodies and immunoblotted with anti-Snail and anti-UBR5 antibodies. (D) Co-localization of UBR5 and Snail in the nucleus. Immunofluorescence assay probe co-localization of UBR5 (red) and Snail (green). Scale bar: 50 μm. (E) UBR5 interacted with Snail through the HECT domain. A schematic of various UBR5 truncations that are fused to His. Coomassie blue staining image of a PAGE gel, confirming the expression of pET28a and various UBR5 truncations. (F) Snail interacted with UBR5 through the zinc-figure domain. A schematic of various Snail truncations that are fused to His. Coomassie blue staining image of a PAGE gel, confirming the expression of pET28a and various Snail truncations. (G) Molecular docking of Snail zinc-figure domain (amino acids 151–264) and UBR5 HECT domain (amino acids 2453–2799) truncation protein. (H) The HECT domain of UBR5 interacted with Snail in the co-immunoprecipitation assay. HEK293T cells were transfected with wild-type and truncated Myc-tagged UBR5, as well as Flag-tagged Snail, and treated with MG132 as indicated. Cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibodies and immunoblotted with anti-Snail or anti-UBR5 antibodies.

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 interacted with Snail. (A) UBR5 was positively correlated with Snail in colon adenocarcinoma and rectal adenocarcinoma. Correlation analysis for TCGA and GTEx on the GEPIA website showed a correlation coefficient of R = 0.26, P = 4.4e-13. P < 0.01 denoted statistical significance. (B) The expression levels of UBR5 and Snail correlated with colorectal cancer (CRC) stages. The UBR5 and Snail mRNA levels based on pathological stages were analyzed using the GEPIA2 violin-plots in colorectal tumors. (C) Co-immunoprecipitation assay showed that UBR5 interacted with Snail. HEK293T cells were transfected with Myc-tagged UBR5 and Flag-tagged Snail and treated with MG132 as indicated. Cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibodies and immunoblotted with anti-Snail and anti-UBR5 antibodies. (D) Co-localization of UBR5 and Snail in the nucleus. Immunofluorescence assay probe co-localization of UBR5 (red) and Snail (green). Scale bar: 50 μm. (E) UBR5 interacted with Snail through the HECT domain. A schematic of various UBR5 truncations that are fused to His. Coomassie blue staining image of a PAGE gel, confirming the expression of pET28a and various UBR5 truncations. (F) Snail interacted with UBR5 through the zinc-figure domain. A schematic of various Snail truncations that are fused to His. Coomassie blue staining image of a PAGE gel, confirming the expression of pET28a and various Snail truncations. (G) Molecular docking of Snail zinc-figure domain (amino acids 151–264) and UBR5 HECT domain (amino acids 2453–2799) truncation protein. (H) The HECT domain of UBR5 interacted with Snail in the co-immunoprecipitation assay. HEK293T cells were transfected with wild-type and truncated Myc-tagged UBR5, as well as Flag-tagged Snail, and treated with MG132 as indicated. Cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibodies and immunoblotted with anti-Snail or anti-UBR5 antibodies.

Article Snippet: The primary antibodies used in this experiment were UBR5 (Proteintech, 66937-1-Ig), Snail (Cell Signaling Technology, Danvers, USA, 3879S), and E-cadherin (Proteintech, 20874-1-AP).

Techniques: Expressing, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Immunofluorescence, Staining

UBR5 promoted the degradation and polyubiquitination of Snail. (A) UBR5 promoted the proteasomal degradation of Snail. HEK293T cells were transfected with Snail-Flag, Snail 6SA-Flag, UBR5-Myc, GFP, or empty vector and treated with DMSO, chloroquine, MG132, or CT99021 as indicated. The expression of Snail and GFP was assessed by western blotting. (B) UBR5 degraded Snail protein in a concentration-dependent manner. HEK293T cells were transfected with Snail-Flag, GFP, or in combination with different concentrations of wild-type and truncated UBR5-Myc for 48 h. Cell lysates were immunoblotted with anti-Snail antibodies. (C) UBR5 promoted K48 polyubiquitinated chain generation of Snail protein. In cellular ubiquitination assays, UBR5-Myc were co-transfected with Snail-Flag plasmids or with HA-Ub-K63 and HA-Ub-K48 plasmids. Western blotting was performed on cell lysates immunoprecipitated with an anti-Flag antibody, followed by the detection of polyubiquitination levels using an anti-Ub antibody. (D) UBR5 accelerated the Snail protein turnover through the HECT domain. HEK293T cells were transfected with corresponding plasmids. Cells were treated with cycloheximide (CHX) and harvested at indicated time points for immunoblotting with anti-Snail or anti-GFP antibody. The graph shows the quantification of Snail protein levels (based on the band intensity from the gels) normalized to those of GFP over the time course. Snail protein expression at the 0 h time point of treatment with CHX was set as 100 %. Experiments were performed in triplicate, and a representative experiment is presented.

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 promoted the degradation and polyubiquitination of Snail. (A) UBR5 promoted the proteasomal degradation of Snail. HEK293T cells were transfected with Snail-Flag, Snail 6SA-Flag, UBR5-Myc, GFP, or empty vector and treated with DMSO, chloroquine, MG132, or CT99021 as indicated. The expression of Snail and GFP was assessed by western blotting. (B) UBR5 degraded Snail protein in a concentration-dependent manner. HEK293T cells were transfected with Snail-Flag, GFP, or in combination with different concentrations of wild-type and truncated UBR5-Myc for 48 h. Cell lysates were immunoblotted with anti-Snail antibodies. (C) UBR5 promoted K48 polyubiquitinated chain generation of Snail protein. In cellular ubiquitination assays, UBR5-Myc were co-transfected with Snail-Flag plasmids or with HA-Ub-K63 and HA-Ub-K48 plasmids. Western blotting was performed on cell lysates immunoprecipitated with an anti-Flag antibody, followed by the detection of polyubiquitination levels using an anti-Ub antibody. (D) UBR5 accelerated the Snail protein turnover through the HECT domain. HEK293T cells were transfected with corresponding plasmids. Cells were treated with cycloheximide (CHX) and harvested at indicated time points for immunoblotting with anti-Snail or anti-GFP antibody. The graph shows the quantification of Snail protein levels (based on the band intensity from the gels) normalized to those of GFP over the time course. Snail protein expression at the 0 h time point of treatment with CHX was set as 100 %. Experiments were performed in triplicate, and a representative experiment is presented.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Concentration Assay, Ubiquitin Proteomics, Immunoprecipitation

UBR5 affected the expression of epithelial–mesenchymal transition (EMT)-related factors. (A) Endogenous UBR5 knockdown changed the expression of Snail and EMT marker genes in colorectal cancer cells. Cells were collected and subjected to immunoblotting analysis and quantitative reverse transcription PCR analysis for indicated epithelial and mesenchymal markers. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (B) Immunofluorescence analysis of Snail and E-cadherin protein expression in control and shUBR5 of HCT116 cells (Snail, green; E-cadherin, red; DAPI, blue). Scale bar: 50 μm. (C) Depletion of UBR5 induced the EMT phenotype in colorectal cancer cells. Morphology of HCT116 cells after transfection with lentiviral shRNAs targeting either control or UBR5. Scale bar: 100 μm. (D) Reduction of UBR5 enhanced cell migration in vitro . Wound-healing experiments were performed to analyze changes in the migratory capacity of HCT116 control and shUBR5 cells. The histogram shows the quantitation of the relative degree of healing ( n = 3). Scale bars: 100 μm. (E) Depletion of UBR5 facilitated cell invasiveness in vitro . Transwell assay was used to analyze changes in the invasive capacity of HCT116 control and shUBR5 cells. Scale bar: 100 μm. The number of cells crossing the basement membrane was counted. The histogram shows the quantitation of the relative numbers of cells that invaded and migrated through the matrix layer ( n = 3). Scale bars: 100 μm. (F) Knockdown of UBR5 increased tumor volumes and weights. The photographs show the excised tumors from HCT116 control (left) and HCT116 shUBR5 (right) models ( n = 3). The tumor sizes (tumor volumes and weights) were subjected to comparison. ∗ P < 0.05 and ∗∗∗ P < 0.001. (G) The knockdown of UBR5 promoted tumor cell infiltration. The effect on the xenograft model in HCT116 control and shUBR5 cells was assessed by hematoxylin-eosin staining. Scale bars: 50 μm.

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 affected the expression of epithelial–mesenchymal transition (EMT)-related factors. (A) Endogenous UBR5 knockdown changed the expression of Snail and EMT marker genes in colorectal cancer cells. Cells were collected and subjected to immunoblotting analysis and quantitative reverse transcription PCR analysis for indicated epithelial and mesenchymal markers. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (B) Immunofluorescence analysis of Snail and E-cadherin protein expression in control and shUBR5 of HCT116 cells (Snail, green; E-cadherin, red; DAPI, blue). Scale bar: 50 μm. (C) Depletion of UBR5 induced the EMT phenotype in colorectal cancer cells. Morphology of HCT116 cells after transfection with lentiviral shRNAs targeting either control or UBR5. Scale bar: 100 μm. (D) Reduction of UBR5 enhanced cell migration in vitro . Wound-healing experiments were performed to analyze changes in the migratory capacity of HCT116 control and shUBR5 cells. The histogram shows the quantitation of the relative degree of healing ( n = 3). Scale bars: 100 μm. (E) Depletion of UBR5 facilitated cell invasiveness in vitro . Transwell assay was used to analyze changes in the invasive capacity of HCT116 control and shUBR5 cells. Scale bar: 100 μm. The number of cells crossing the basement membrane was counted. The histogram shows the quantitation of the relative numbers of cells that invaded and migrated through the matrix layer ( n = 3). Scale bars: 100 μm. (F) Knockdown of UBR5 increased tumor volumes and weights. The photographs show the excised tumors from HCT116 control (left) and HCT116 shUBR5 (right) models ( n = 3). The tumor sizes (tumor volumes and weights) were subjected to comparison. ∗ P < 0.05 and ∗∗∗ P < 0.001. (G) The knockdown of UBR5 promoted tumor cell infiltration. The effect on the xenograft model in HCT116 control and shUBR5 cells was assessed by hematoxylin-eosin staining. Scale bars: 50 μm.

Techniques: Expressing, Knockdown, Marker, Western Blot, Reverse Transcription, Immunofluorescence, Control, Transfection, Migration, In Vitro, Quantitation Assay, Transwell Assay, Membrane, Comparison, Staining

UBR5 C2768S mutation abrogated the interaction with Snail. (A) His pull-down assays showed the abolished interactions between Snail and the UBR5 C2768S. A schematic representation of the UBR5 wild-type and C2768S mutation. (B) Co-immunoprecipitation assay showed that the interaction between the Snail and the UBR5 C2768S mutation was eliminated. HEK293T cells were transfected with UBR5-Myc, UBR5 C2768S-Myc, and Snail-Flag as indicated. Cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibodies and immunoblotted with anti-Snail and anti-UBR5 antibodies. (C) UBR5 C2768S abolished the UBR5-mediated degradation of Snail. HEK293T cells were transfected with Snail-Flag, UBR5-Myc, and UBR5 C2768S-Myc as indicated. Cell lysates were subjected to western blotting analysis with anti-Snail and anti-GFP antibodies. (D) UBR5 C2768S did not accelerate Snail protein turnover. HEK293T cells were transfected with Snail-Flag, UBR5-Myc, and UBR5 C2768S-Myc and treated with cycloheximide (CHX) as indicated. Cell lysates were subjected to western blotting analysis with anti-Snail and anti-GFP antibodi.

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 C2768S mutation abrogated the interaction with Snail. (A) His pull-down assays showed the abolished interactions between Snail and the UBR5 C2768S. A schematic representation of the UBR5 wild-type and C2768S mutation. (B) Co-immunoprecipitation assay showed that the interaction between the Snail and the UBR5 C2768S mutation was eliminated. HEK293T cells were transfected with UBR5-Myc, UBR5 C2768S-Myc, and Snail-Flag as indicated. Cell lysates were immunoprecipitated with either anti-Myc or anti-Flag antibodies and immunoblotted with anti-Snail and anti-UBR5 antibodies. (C) UBR5 C2768S abolished the UBR5-mediated degradation of Snail. HEK293T cells were transfected with Snail-Flag, UBR5-Myc, and UBR5 C2768S-Myc as indicated. Cell lysates were subjected to western blotting analysis with anti-Snail and anti-GFP antibodies. (D) UBR5 C2768S did not accelerate Snail protein turnover. HEK293T cells were transfected with Snail-Flag, UBR5-Myc, and UBR5 C2768S-Myc and treated with cycloheximide (CHX) as indicated. Cell lysates were subjected to western blotting analysis with anti-Snail and anti-GFP antibodi.

Techniques: Mutagenesis, Co-Immunoprecipitation Assay, Transfection, Immunoprecipitation, Western Blot

C2768S mutation abolished the effects of UBR5 on the migration and invasion of HCT116 cells. (A) C2768S mutation changed the expression of epithelial–mesenchymal transition marker genes. HCT116 cells were transfected with UBR5-Myc and UBR5 C2768S-Myc constructs. Cells were collected and subjected to immunoblotting analysis and quantitative reverse transcription PCR analysis for indicated epithelial and mesenchymal markers. ∗∗ P < 0.01 and ∗∗∗ P < 0.001. (B) Immunofluorescence analysis of Snail and E-cadherin protein expression in Mock, UBR5-Myc, and UBR5 C2768S-Myc in HCT116 cells (Snail, green; E-cadherin, red; DAPI, blue). Scale bar: 50 μm. (C) Wound-healing assays showed the migration of HCT116 cells transfected with Mock, UBR5-Myc, or UBR5 C2768S-Myc. Representative images of healing degrees at 0 and 48 h after performing the wound are shown. The histogram shows the quantitation of the relative healing degrees ( n = 3). Scale bars: 100 μm. (D) Transwell assays showed the invasiveness of HCT116 cells transfected with Mock, UBR5-Myc, or UBR5 C2768S-Myc. Representative images of the staining of the cells that invaded and migrated through the matrix layer are shown. The histogram shows the quantitation of the relative numbers of cells that invaded and migrated through the matrix layer ( n = 3). Scale bars: 100 μm. (E) Wild-type UBR5 tumors were smaller in volume and weight than the HCT116 Mock and C2768S mutant groups. The photographs show the excised tumors from HCT116 Mock, UBR5, and UBR5 C2768S models ( n = 4). The tumor sizes (tumor volumes and weights) were subjected to comparison. (F) The UBR5 C2768S mutation disrupted the UBR5-Snail axis, eliminating its regulatory effect on tumor cell invasion. Hematoxylin-eosin staining of xenograft tumors derived from HCT116 Mock, UBR5-Myc, and UBR5 C2768S-Myc cells. Scale bar: 50 μm.

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: C2768S mutation abolished the effects of UBR5 on the migration and invasion of HCT116 cells. (A) C2768S mutation changed the expression of epithelial–mesenchymal transition marker genes. HCT116 cells were transfected with UBR5-Myc and UBR5 C2768S-Myc constructs. Cells were collected and subjected to immunoblotting analysis and quantitative reverse transcription PCR analysis for indicated epithelial and mesenchymal markers. ∗∗ P < 0.01 and ∗∗∗ P < 0.001. (B) Immunofluorescence analysis of Snail and E-cadherin protein expression in Mock, UBR5-Myc, and UBR5 C2768S-Myc in HCT116 cells (Snail, green; E-cadherin, red; DAPI, blue). Scale bar: 50 μm. (C) Wound-healing assays showed the migration of HCT116 cells transfected with Mock, UBR5-Myc, or UBR5 C2768S-Myc. Representative images of healing degrees at 0 and 48 h after performing the wound are shown. The histogram shows the quantitation of the relative healing degrees ( n = 3). Scale bars: 100 μm. (D) Transwell assays showed the invasiveness of HCT116 cells transfected with Mock, UBR5-Myc, or UBR5 C2768S-Myc. Representative images of the staining of the cells that invaded and migrated through the matrix layer are shown. The histogram shows the quantitation of the relative numbers of cells that invaded and migrated through the matrix layer ( n = 3). Scale bars: 100 μm. (E) Wild-type UBR5 tumors were smaller in volume and weight than the HCT116 Mock and C2768S mutant groups. The photographs show the excised tumors from HCT116 Mock, UBR5, and UBR5 C2768S models ( n = 4). The tumor sizes (tumor volumes and weights) were subjected to comparison. (F) The UBR5 C2768S mutation disrupted the UBR5-Snail axis, eliminating its regulatory effect on tumor cell invasion. Hematoxylin-eosin staining of xenograft tumors derived from HCT116 Mock, UBR5-Myc, and UBR5 C2768S-Myc cells. Scale bar: 50 μm.

Techniques: Mutagenesis, Migration, Expressing, Marker, Transfection, Construct, Western Blot, Reverse Transcription, Immunofluorescence, Quantitation Assay, Staining, Comparison, Derivative Assay

UBR5 was a favorable prognostic factor in human colorectal cancer. (A) Snail was highly expressed in samples obtained from patients with colorectal cancer. Immunohistochemical analysis of Snail expression levels in normal colorectum and tumors in the Human Protein Atlas website. Representative images are shown. ∗∗∗ P < 0.001; student's t -test. (B) GEPIA revealed that UBR5 had higher expression in normal tissue samples compared with tumor samples. Dark and light gray indicate tumor and normal tissues, respectively. (C) High expression of UBR5 was associated with a favorable prognosis. Kaplan–Meier analysis of 20-year overall survival of rectum adenocarcinoma cancer patients with UBR5 ( n = 165). Log-rank P -values are shown. (D) Diagram of the pattern of UBR5 regulation of epithelial–mesenchymal transition (EMT) in colorectal cancer. UBR5 inhibited the invasive migration of tumor cells by regulating the ubiquitination and transcriptional activity of Snail, and UBR5 C2768S eliminated the inhibitory effect of UBR5 on EMT.

Journal: Genes & Diseases

Article Title: UBR5 regulates the progression of colorectal cancer cells through Snail-induced epithelial–mesenchymal transition

doi: 10.1016/j.gendis.2025.101679

Figure Lengend Snippet: UBR5 was a favorable prognostic factor in human colorectal cancer. (A) Snail was highly expressed in samples obtained from patients with colorectal cancer. Immunohistochemical analysis of Snail expression levels in normal colorectum and tumors in the Human Protein Atlas website. Representative images are shown. ∗∗∗ P < 0.001; student's t -test. (B) GEPIA revealed that UBR5 had higher expression in normal tissue samples compared with tumor samples. Dark and light gray indicate tumor and normal tissues, respectively. (C) High expression of UBR5 was associated with a favorable prognosis. Kaplan–Meier analysis of 20-year overall survival of rectum adenocarcinoma cancer patients with UBR5 ( n = 165). Log-rank P -values are shown. (D) Diagram of the pattern of UBR5 regulation of epithelial–mesenchymal transition (EMT) in colorectal cancer. UBR5 inhibited the invasive migration of tumor cells by regulating the ubiquitination and transcriptional activity of Snail, and UBR5 C2768S eliminated the inhibitory effect of UBR5 on EMT.

Techniques: Immunohistochemical staining, Expressing, Migration, Ubiquitin Proteomics, Activity Assay

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) Knockdown of MARCHF7 or UBR5 resulted in nsp16 restoration. HEK293T cells were transfected with small interfering RNA (siRNA) of E3 ligase candidates for 24 hr, followed by co-incubation with the nsp16-Flag-expressing plasmids for 48 hr, treated with MG132 (10 µM) for 16 hr before harvesting, lysed, and subjected to immunoblotting (IB) assay using anti-Flag antibody. RT-qPCR was conducted to determine the mRNA expression levels of E3 ligase candidates. The siRNA targeting regions for the candidate E3 ubiquitin ligase proteins and the targeted regions for RT-qPCR are shown in . Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( B ) RNA levels of UBR5 or MARCHF7 from HEK293T cells infected with lentivirus containing control or shRNA targeting UBR5 or MARCHF7 for 48 hr and screened with antibiotics for 48 hr. Knockdown cell lines were transfected with plasmids expressing nsp16-Flag, collected at the indicated times, and the protein levels of nsp16, MARCHF7, and UBR5 were detected by IB. ( C ) MARCHF7 and UBR5 acted separately and did not depend on each other. HEK293T cells stably expressing UBR5 shRNA or MARCHF7 shRNA were transfected with siRNA of MARCHF7 or UBR5 for 24 hr, respectively, followed by co-incubation with the nsp16-Flag-expressing plasmids for 48 hr. The protein levels and the RNA levels of nsp16, UBR5, and MARCHF7 were measured by IB and RT-qPCR, respectively. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( D, E ) In HEK293T cells stably expressing UBR5 shRNA or MARCHF7 shRNA, nsp16 was degraded by overexpressed UBR5 or MARCHF7, respectively, whereas the mutant failed to degrade nsp16. The cell lysates were analyzed by anti-Flag antibody. Figure 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—source data 2. Original files for western blot analysis displayed in . Figure 2—source data 3. Numerical data obtained during experiments represented in .

Article Snippet: UBR5 (Gene ID: 51366) and its mutants (UBR5-ΔHECT, UBR5-ΔPABC, UBR5-ΔUBR) expression plasmids were constructed using purchased plasmids from Addgene (Watertown, MA, USA) as templates with no tag or a MYC tag at the N-terminus.

Techniques: Knockdown, Transfection, Small Interfering RNA, Incubation, Expressing, Western Blot, Quantitative RT-PCR, Ubiquitin Proteomics, Two Tailed Test, Infection, Control, shRNA, Stable Transfection, Mutagenesis

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A, B ) UBR5 small interfering RNA (siRNA) was transfected into shMARCHF7 cells to knock down UBR5 . After 24 hr, MARCHF7 and nsp16 expression vectors were co-transfected, and the cells were harvested 72 hr later. The levels of nsp16 were characterized by immunoblotting (IB) with anti-Flag antibody. Whether MARCHF7 was dependent on UBR5 to degrade nsp16 was determined by further transfection of MARCHF7 siRNA into shUBR5 cells, followed by co-transfection of UBR5 and nsp16 expression vectors 24 hr later. The other operations are the same as above. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test. p>0.05; **p<0.01; ***p<0.001. ( C ) The siRNA targeting regions and RT-qPCR targeting regions for the E3 ubiquitin ligases—HECTD1, UBR5, MYCBP2, TRIM21, TRIM32, and MARCHF7—are shown. Figure 2—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 2—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 2—figure supplement 1—source data 3. Numerical data obtained during experiments represented in .

Techniques: Small Interfering RNA, Transfection, Knockdown, Expressing, Western Blot, Cotransfection, Two Tailed Test, Quantitative RT-PCR, Ubiquitin Proteomics

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) Nsp16 can be ubiquitinated. HEK293T cells co-transfected with ubiquitin-Myc and nsp16-Flag or transfected with nsp16-Flag alone. The cells were treated with MG132 for 12 hr before collection. The whole-cell lysates were incubated with anti-Flag beads and used for immunoblotting (IB) with anti-Myc or anti-Flag antibodies to detect the polyubiquitination chain of nsp16. ( B ) Assess the endogenous ubiquitination level of nsp16 protein. Cells were transfected with nsp16-Flag or an empty vector and collected 48 hr later. Prior to harvesting, cells were treated with MG132 for 16 hr. Co-immunoprecipitation (Co-IP) experiments were then performed to analyze the endogenous ubiquitination level of nsp16. ( C ) The level of ubiquitination of nsp16 decreased with decreasing the protein levels of MARCHF7 or UBR5. E3 was knocked down by transfection with small interfering RNA (siRNA) targeting UBR5 or MARCHF7 , and 24 hr later, ubiquitin-Myc and nsp16-HA were co-transfected or nsp16-HA alone. Cells were treated with MG132 for 16 hr before collection. Whole-cell lysates were incubated with anti-HA beads, and polyubiquitinated chains of nsp16 were detected by IB with anti-Myc or anti-HA antibodies. ( D ) Nsp16 can be modified by a variety of ubiquitin chains. HEK293T cells were transfected with either nsp16-HA alone or together with plasmids encoding various mutants of ubiquitin (K6 only, K11 only, K27 only, K29 only, K33 only, K48 only, K63 only). Thirty-six hours later, cells were treated with MG132 for 12 hr. Cell lysates were then subjected to immunoprecipitation, followed by IB to analysis. ( E, F ) MARCHF7 or UBR5 causes nsp16 to be modified by the K27-type or K48-type ubiquitin chain. 293T cell lines with or without MARCHF7 or UBR5 knockdown were co-transfected with plasmids encoding ubiquitin-WT or various mutants of ubiquitin (K6 only, K11 only, K27 only, K29 only, K33 only, K48 only, K63 only). The other experimental methods were the same as C. Figure 3—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 3—source data 2. Original files for western blot analysis displayed in .

Techniques: Transfection, Ubiquitin Proteomics, Incubation, Western Blot, Plasmid Preparation, Immunoprecipitation, Co-Immunoprecipitation Assay, Small Interfering RNA, Modification, Knockdown

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) HEK293T cells were transfected with either nsp16-Flag alone or together with MARCHF7-Myc. Thirty-six hours after transfection, the cells were treated with MG132 (10 µM) for 12 hr. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody. Using immunoblotting (IB) to analyze the precipitates and input. ( B ) HEK293T cells were transfected with nsp16-Flag. Cell lysates were subjected to immunoprecipitation with anti-UBR5 or IgG antibody. ( C, D ) HeLa cells were co-transfected with YFP-nsp16 and CFP-UBR5 or CFP-MARCHF7. A representative image of YFP-nsp16 (yellow) and ECFP-MARCHF7 (cyan) or ECFP-UBR5 (cyan) expressing cells before and after photobleaching the acceptor fluorophore, YFP. The region chosen for photobleaching is marked (white open box). Scale bars, 10 µm. The quantization of fluorescence brightness was analyzed by ImageJ. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test. p>0.05; **p<0.01; ***p<0.001. ( E ) HeLa cells transfected with nsp16-Flag were analyzed by confocal microscopy. The Flag-tagged nsp16 labeled with anti-Flag antibody (red). MARCHF7 or UBR5 were labeled with endogenous antibodies (green). Cell nuclei were stained using DAPI (4′,6-diamidino-2-phenylindole) (blue). Representative images were shown. Scale bars, 20 µm. The ratio of colocalization was quantified by measuring the fluorescence intensities using ImageJ. ( F ) nsp16 was stably transfected into HEK293T cells. The cells were analyzed by confocal microscopy. The other operations are the same as above. Figure 4—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 4—figure supplement 1—source data 3. Numerical data obtained during experiments represented in .

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Fluorescence, Two Tailed Test, Confocal Microscopy, Labeling, Staining, Stable Transfection

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A, B ) The binding of MARCHF7 or UBR5 to nsp16 was not mutually dependent. The binding of nsp16 to UBR5 or MARCHF7 was identified by co-immunoprecipitation in HEK293T cells transfected into si MARCHF7 or si UBR5 , respectively. The immunoprecipitates and input were analyzed by immunoblotting (IB). The knockdown efficiency was detected by RT-qPCR and IB. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by a two-tailed t-test: ***p<0.001. ( C, D ) MARCHF7 or UBR5 colocalized with nsp16 in the endoplasmic reticulum. Hela cells were co-transfected with YFP-nsp16 (yellow) and CFP-UBR5 (cyan) or CFP-MARCHF7 (cyan). The organelles were labeled with antibodies against marker proteins of endoplasmic reticulum, Golgi apparatus, and mitochondria respectively (red). The cells were analyzed by confocal microscopy ( C ). Scale bars, 20 µm. The ratio of colocalization was quantified by measuring the fluorescence intensities using ImageJ ( D ). Figure 4—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—source data 2. Original files for western blot analysis displayed in . Figure 4—source data 3. Numerical data obtained during experiments represented in .

Techniques: Binding Assay, Immunoprecipitation, Transfection, Western Blot, Knockdown, Quantitative RT-PCR, Two Tailed Test, Labeling, Marker, Confocal Microscopy, Fluorescence

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) The schematic represents UBR5 wild-type (WT) or mutants used in the study. ( B ) The homologous to the E6AP carboxyl terminus (HECT) domain of UBR5 is required for nsp16 degradation. After co-transfection with UBR5 WT or mutants and nsp16-HA, cells were harvested 48 hr later, and cell lysates were examined by immunoblotting (IB). ( C ) The HECT domain of UBR5 affects K48-type ubiquitin chain of nsp16. HEK293T cells were transfected with the assigned plasmids. After 36 hr, cells were treated with 10 µM MG132 for 12 hr, harvested, and cell lysates were incubated with protein G agarose beads conjugated with anti-HA antibodies. Cell lysates and precipitated samples were analyzed by IB. ( D ) The schematic represents WT and truncated forms of MARCHF7 used in the study. (E) Only MARCHF7 WT degraded nsp16. ( F ) The N-terminal region of MARCHF7 interacted with nsp16, and only the WT could catalyze the K27-type ubiquitin chain of nsp16. Figure 4—figure supplement 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 4—figure supplement 2—source data 2. Original files for western blot analysis displayed in .

Techniques: Cotransfection, Western Blot, Ubiquitin Proteomics, Transfection, Incubation

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A, B ) Knocking down MARCHF7 or UBR5 enhances SARS-CoV-2 trVLP infectivity. MARCHF7 or UBR5 was knocked down by small interfering RNA (siRNA) in Caco2 cells with stable expression of SARS-CoV-2 N protein. Twenty-four hours later, cells were infected with SARS-CoV-2 virus-like particles (MOI: 0.1), the medium was changed 2 hr after infection, and the eGFP-positive cells were detected by flow cytometry 48 hr later ( A ). Protein content was determined by RT-qPCR ( B ). Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest: *p<0.05; **p<0.01; ***p<0.001. Figure 5—figure supplement 1—source data 1. Numerical data obtained during experiments represented in .

Techniques: Infection, Small Interfering RNA, Expressing, Virus, Flow Cytometry, Quantitative RT-PCR

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) The virus-encoded nsp16 protein interacts with endogenous MARCHF7 and UBR5 and undergoes ubiquitination modification. In 293T-ACE2 cells, with or without IME-BJ01 strain infection (MOI: 0.01), the medium was changed 2 hr post-infection, and cells were harvested 48 hr later, with MG132 treatment added 16 hr before harvesting. nsp16 protein was enriched using protein G beads coupled with the nsp16 antibody, and interactions and ubiquitination were analyzed by immunoblotting (IB) with endogenous antibodies against MARCHF7, UBR5, and ubiquitination. ( B–I ) MARCHF7 and UBR5 were knocked down by small interfering RNA (siRNA) in Caco2 cells. 24 hr after transfection, the cells were infected with IME-BJ01 strain (MOI: 0.01) ( C–E ) or Omicron BA.1 strain (MOI: 0.001) ( F–H ), respectively. 2 hr post-infection, the supernatant was discarded, and the cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 3% fetal bovine serum for 48 hr. The mRNA levels of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) M and E genes in the cells ( C, F ) and E genes in supernatant ( D, G ) were detected by RT-qPCR, and the viral titers in supernatant ( E, H ) were measured. The N protein levels of IME-BJ01 or Omicron viruses were detected by IB ( I ). Knockdown efficiencies of MARCHF7 and UBR5 were detected by RT-qPCR or IB ( B, I ). Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest: *p<0.05; **p<0.01; ***p<0.001. Figure 5—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 5—source data 2. Original files for western blot analysis displayed in . Figure 5—source data 3. Numerical data obtained during experiments represented in .

Techniques: Virus, Ubiquitin Proteomics, Modification, Infection, Western Blot, Small Interfering RNA, Transfection, Cell Culture, Quantitative RT-PCR, Knockdown

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A–H ) UBR5 or MARCHF7 was transfected in 293T cells stably overexpressed with ACE2, and the increased doses of nsp16-Flag were transfected simultaneously. After 24 hr, the cells were infected with IME-BJ01 strains. The mRNA levels of M and E genes of the IME-BJ01 strain in the cells ( A, D ) and E gene in supernatant ( B, E ) were detected by RT-qPCR, as well as the detection of viral titers in supernatant ( C, F ). The N protein of the virus and the overexpression efficiency were detected by IB ( G, H ). Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. p>0.05; **p<0.01; ***p<0.001. shows data related to infection with Omicron BA.1. Figure 6—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—source data 2. Original files for western blot analysis displayed in . Figure 6—source data 3. Numerical data obtained during experiments represented in .

Techniques: Transfection, Stable Transfection, Infection, Quantitative RT-PCR, Virus, Over Expression, Western Blot

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A–H ) In 293T-ACE2 cells, the Really Interesting New Gene (RING) domain deletion mutant of MARCHF7 (MARCHF7-aa 1–542) or the homologous to the E6AP carboxyl terminus (HECT) domain inactivated mutant of UBR5 (UBR5-ΔHECT) were transfected, along with a gradient of nsp16-Flag overexpression. The cells were infected with the IME-BJ01 strain (MOI: 0.01), medium was changed 2 hr post-infection, and cells and supernatants were collected 48 hr after infection. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. p>0.05; **p<0.01; ***p<0.001. Figure 6—figure supplement 2—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 2—source data 2. Original files for western blot analysis displayed in . Figure 6—figure supplement 2—source data 3. Numerical data obtained during experiments represented in .

Techniques: Mutagenesis, Transfection, Over Expression, Infection, Western Blot

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) This diagram shows the mutation of nsp16 in different virus subtypes. The amino acid sequences of different SARS-CoV-2 strains were obtained from the National Center for Biotechnology Information (NCBI), and the amino acid sequences of nsp16 of different strains were compared by DNAMAN software. ( B ) Nsp16 mutants can still be regulated by MG132. The mutated nsp16 plasmids were transfected into HEK293T cells. After 36 hr of culture, cells were treated with 10 µm MG132 or DMSO, harvested 12 hr later, and cell lysates were examined by immunoblotting (IB). ( C ) MARCHF7 or UBR5 can degrade nsp16 mutants. After transfecting MARCHF7 or UBR5 small interfering RNA (siRNA) and the mutated nsp16 plasmids, the cells were harvested 48 hr later. The cell lysates were detected by IB. Figure 6—figure supplement 3—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 3—source data 2. Original files for western blot analysis displayed in .

Techniques: Mutagenesis, Virus, Software, Transfection, Western Blot, Small Interfering RNA

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A–C ) The protein and mRNA levels of MARCHF7 or UBR5 upon infection with different titers. Endogenous MARCHF7 and UBR5 RNA levels were detected by RT-qPCR 48 hr after infection with different titers of IME-BJ01 strain (MOI: 0, 0.0001, 0.001, 0.01) or Omicron BA.1 strain (MOI: 0, 0.0001, 0.001). Protein levels were examined by immunoblotting (IB). ( D ) The expression level of UBR5 was negatively correlated with the severity of the disease, but MARCHF7 expression levels were not. Peripheral blood mononuclear cells (PBMCs) were extracted from common, severe, and critical coronavirus disease 2019 (COVID-19) patients. RT-qPCR was used to detect the mRNA level of UBR5 or MARCHF7 in patients. Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. ns, p>0.05; **p<0.01; ***p<0.001. Figure 6—figure supplement 4—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 6—figure supplement 4—source data 2. Original files for western blot analysis displayed in . Figure 6—figure supplement 4—source data 3. Numerical data obtained during experiments represented in .

Techniques: Infection, Quantitative RT-PCR, Western Blot, Expressing

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A–G ) BLAB/C mice were injected with the corresponding plasmids at 40 µg/500 µl via the high-pressure tail vein, followed by nasal inoculation with 50 µl severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus at a dosage of 10 5.5 TCID50/ml (created with BioRender.com and the agreement no. is OO281XWHNA). Immunoblotting (IB) was used to detect the expression of MARCHF7 or UBR5 in the lung tissues ( B ). Viral RNA loads in mouse lung tissues were detected by measuring the mRNA levels of the E genes by RT-qPCR ( C ). Lung tissue was collected, homogenized, and the residue was removed by centrifugation to collect the supernatant. The viral titer was then measured using the TCID50 method ( D ). Mouse body weight was monitored during the experimental period ( E ). Representative images of hematoxylin and eosin (H&E) staining of lungs of mice with different treatments. Magnification, ×40. Scale bars, 20 µm ( F ). The staining of viral N proteins. Magnification, ×63. Scale bars, 20 µm. n=3 in each group ( G ). RT-qPCR was used to measure the expression of cytokines and chemokines in the spleens of mice in each group ( H ). Statistical significance was analyzed using a one-way analysis of variance with Tukey’s multiple comparisons test (NS, no significance, *p<0.05, **p<0.01, ***p<0.001). Figure 7—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 7—source data 2. Original files for western blot analysis displayed in . Figure 7—source data 3. Numerical data obtained during experiments represented in .

Techniques: Injection, Virus, Western Blot, Expressing, Quantitative RT-PCR, Residue, Centrifugation, Staining

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: Schematic diagram of MARCHF7 and UBR5 ubiquitinate the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein nsp16, leading to its degradation via the proteasomal pathway, thereby affecting viral replication (created with BioRender.com and the agreement no. is EV281XWATL).

Techniques:

Journal: eLife

Article Title: SARS-CoV-2 nsp16 is regulated by host E3 ubiquitin ligases, UBR5 and MARCHF7

doi: 10.7554/eLife.102277

Figure Lengend Snippet: ( A ) Single-lysine mutation of nsp16 protein can be restored by MG132. Single-lysine mutants of nsp16 protein were obtained through mutagenesis and overexpressed in 293T cells. After 36 hr, cells were treated with either MG132 or DMSO for 16 hr and then harvested. Protein levels were detected by immunoblotting (IB) using the HA antibody. ( B ) nsp16 protein truncates can be restored by MG132. nsp16 protein truncates were obtained through structural analysis and mutations. IB was performed to determine whether the truncates could be restored by MG132. ( C ) The mass spectrometry analysis identified the ubiquitination modification site at lysine 76. nsp16-Flag was overexpressed in 293T cells, followed by MG132 treatment and cell harvest. nsp16 protein was enriched using Flag antibody-conjugated protein G beads. Flag-peptide competition was used to obtain the nsp16-containing solution. The protein and ubiquitination status were visualized by SDS-PAGE and Coomassie staining. Mass spectrometry was used for further analysis. ( D ) Degradation of nsp16-K76R is still regulated by MARCHF7 or UBR5. A plasmid with a mutation at lysine 76 of nsp16 to arginine (nsp16-K76R) was constructed. MARCHF7 or UBR5 was knocked down by small interfering RNA (siRNA) in 293T cells. nsp16-WT or nsp16-K76R was transfected the next day, and cells were harvested 48 hr later. Protein levels were detected by IB. ( E ) Ubiquitination levels of nsp16-K76R are reduced but still regulated by MARCHF7 or UBR5. MARCHF7 or UBR5 was knocked down using siRNA in 293T cells. The cells were co-transfected with Ub-K27 or K48, and nsp16-WT or nsp16-K76R mutant. Cells were harvested 48 hr later, with MG132 treatment 16 hr before harvesting. Co-immunoprecipitation (Co-IP) experiments were performed to analyze the ubiquitination status of nsp16-WT or its mutant. Data are representative of three independent experiments and shown as average ± SD (n=3). Significance was determined by one-way ANOVA, followed by a Tukey’s multiple comparisons posttest. p>0.05; **p<0.01; ***p<0.001. Figure 8—figure supplement 1—source data 1. PDF file containing original western blots for , indicating the relevant bands and treatments. Figure 8—figure supplement 1—source data 2. Original files for western blot analysis displayed in . Figure 8—figure supplement 1—source data 3. Numerical data obtained during experiments represented in .

Techniques: Mutagenesis, Western Blot, Mass Spectrometry, Ubiquitin Proteomics, Modification, SDS Page, Staining, Plasmid Preparation, Construct, Small Interfering RNA, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay

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