EMBO/SourceData huggingface dataset:
Figure 5A RNA degradation is not essential for asymmetric HXT2 mRNA localization. FISH of wild-type and ∆xrn1 cells.B The cytoskeleton contributes to HXT2 mRNA enrichment in the bud. Cells were treated upon glucose addition with either 120 mg/ml benomyl for 15 min, 30 mg/ml latrunculin A for 30 min or 200 µM CK-666 for 30 min. HXT2 mRNA was detect by FISH.C Asymmetric loading of HXT2 mRNA onto the nuclear envelope and enrichment in the bud requires Kar9 but not Bim1 function. HXT2 mRNA FISH with ∆bim1 and ∆kar9 strains. Arrows point to a nucleus with HXT2 signal. D Quantification of the daughter cell enrichment from data displayed in (C). E Quantification of the HXT2 mRNA signal in cells with two nuclei in the mother cell from the experiment depicted in (C). Percentage of cells with asymmetric HXT2 mRNA staining is displayed.
Figure 6A Deletion of NUP2 and MLP1/2 reduced HXT2 mRNA enrichment in the bud. FISH under glucose shift conditions with strains in which different NPC components were deleted. B Quantification of (A). C HXT2 mRNA is on the nuclear envelope after MAT upon glucose shift. FISH-IF experiment with a probe against HXT2 mRNA and an antibody against the HA tag of the nuclear pore complex component Nup84-HA. White arrows point to HXT2 mRNA signals on the nuclear envelope.D HXT2 mRNA travels on the nuclear envelope into the bud during mitosis after glucose shift. Stills of time lapse movies. HXT2 mRNA was visualized using the MS2-GFP system. Under glucose shift conditions HXT2 mRNA is mostly localized to the bud together with the nucleus. Under glucose rich conditions HXT2 mRNA moves mostly independent of the nucleus. Cyan: Nup84-mCherry; white arrowhead: HXT2 mRNA-MS2 + MCP-GFP.
Figure 7HXT2 only strains grow faster than the other HXT only strain. Growth was assessed 2-3 days after incubation at 30°C.D Quantification of competition assay. qPCR with primers against either HXT1, 2, 3 or 4 was performed. HXT gene abundance was normalized against actin.E Strains that lack HXT2 grow slower when coming from quiescence compared to WT.F Tagging the HXT2 mRNA with U1A stem loops leads to its artificial enrichment in the bud in 96% of cells independently of the cell cycle. Arrows point to examples of HXT2-U1A mRNA in the bud early and late in the cell cycle.G Cells that always enrich HXT2 mRNA in the bud have a growth advantage in the first hours when coming from quiescence compared to WT or cells that only express HXT2.H Daughter cells do not retain a memory of the previous stress situation encountered by their mothers. Cells were re-fed after starvation and analyzed for enrichment of HXT2 mRNA via FISH at different time points.
Figure 1A. Cryo-sections of human organoids were analyzed for the presence of enterocytes (E-cad), Goblet cells (Muc-2) and tight junctions (ZO-1) by indirect immunofluorescence. Nuclei are stained with DAPI. Scale bar 25 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.B. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. 16hpi organoids were frozen, cryo-sectioned, and HAstV1 infected cells were visualized by indirect immunofluorescence (HAstV1 (red), nuclei (DAPI, blue). Scale bar 25 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.C. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. Organoids at 16 hpi were fixed and the presence of HAstV1 infected cells (green) were visualized by indirect immunofluorescence. Apical and basolateral membranes were immunostained for actin (magenta) and Laminin (red) respectively. Nuclei are stained with DAPI (blue). Scale bar is 20 μm. Data information: Three biological replicates were performed for each experiment. Representative immunofluorescence images are shown. Error bars indicate the standard deviation. Statistics are from unpaired t-test.E. Human intestinal organoids were infected with HAstV1. At indicated time post-infection the increase in viral copy number was determined by q-RT-PCR.F. Human intestinal organoids were incubated with media (mock) or infected with HAstV1. At 24 hpi the presence of IFNλ in the media was tested by ELISA. Dotted line indicates detection limit of the assay.G. Same as E but for the induced levels of either type I IFN (IFNβ1) or type III IFN (IFNλ).H. Human intestinal organoids were pre-treated for 24h with 2000IU/mL of IFNβ1 or 300ng/mL of IFNλ1-3. Interferons were maintained during the course of infection and the amount of HAstV1 copy numbers was assayed 24hpi by q-RT-PCR.
Figure 3J-M. Percentage of stem cells (OLFM4) goblet cell (FGCBP), mature enterocytes (APOA4) and cells of the enterocyte lineage (FGCBP) from the total organoid cells population (black bar) in mock-treated organoids and after HAstV1 infection. The percentage of infected cells (red bars) and bystander cells (blue bars) within each cell type is shown. The data was obtained from multiplex in situ RNA hybridization in 2D organoids. n=10, mean ± SEM. Statistics show comparison of infected cells between mock-treated, 4h pi and 16h pi organoids. An Ordinary one-way ANOVA and Tukey's multiple comparisons test was used. n.s non significant, * p<0.05, ** p<0.01, *** p<0.001.
Figure 5A. One image per condition was used to show the multiplex in situ RNA FISH. A representative region of the images was chosen and cropped in order to have a zoom-in of a specific area for better visualization. Shown are the enterocyte lineage marker FABP6 (green), HAstV1 infection (white), and CCL2 gene expression (red) of mock-treated and infected organoids. DAPI is in blue. White arrows show co-localization of FABP6 and CCL2 with HAstV1 signal. Scale bar 100µm.B-D. Fluorescence intensity (arbitrary units) of innate immune marker expression in stem cells (OLFM4 positive), goblet cells (FCGBP positive), enterocyte lineage cells (FABP6 positive), and mature enterocytes (APOA4 positive). Bystander cells are in blue and infected cells in red. 10-90 percentile box plots, each dot represents one cell (mock N=2274, 4h N=3682, 16h N=1510. C. mock N=2693, 4h N=3706, 16h N=1921. D. mock N=2274, 4h N=3706, 16h N=1514.) . Blue statistics show a comparison of bystander cells between cell lineages and red statistics show the comparison of infected cells between cell lineages. Ordinary one-way ANOVA and Tukey's multiple comparisons test were used. Grey statistics show the comparison between infected and bystander cells within each cell lineage. Unpaired t-test with Welch's correction was used. n.s non significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001
Figure 1(A) Overexpression of RNF26 promoted polyubiquitination of MITA. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and HA-Ub (1 µg) together with a control or RNF26 expression plasmid (0.5, 1, 2 µg). Twenty-four hours after transfection, cells were subjected to immunoprecipitation (IP) under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-HA (upper panel), anti-Flag (middle panel) and anti-RNF26 (bottom panel). Ub, ubiquitin.(B) RNF26-mediated polyubiquitination of MITA depends on the enzymatic activity of RNF26. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and HA-Ub (1 µg) together with RNF26 or its mutants (1 µg each). IP under denatured conditions and ubiquitination assay was performed as described in (A).(C) UbcH5 mediated polyubiquitination of MITA by RNF26. The RNF26 and MITA proteins were obtained by in vitro transcription and translation, then incubated with biotin-Ub, E1 and the indicated E2s. Polyubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels).(D) RNF26 but not its enzymatic inactive mutants targeted MITA for polyubiquitination in vitro. MITA, RNF26 and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and MITA were incubated with RNF26 or its mutants, followed by ubiquitination and immunoblot analysis as described in (C). All experiments were repeated for at least three times with similar results.
Figure 2(A) RNF26 interacted with MITA. The 293 cells (5×106) were transfected with the indicated plasmids (5 µg each). Twenty-four hours later, cells were lysed and cell lysates were immunoprecipitated with anti-Flag or control IgG. The immunoprecipitates and whole cell lysates were analyzed by immunoblots with anti-HA or anti-Flag.(B) RNF26 was physiologically associated with MITA. THP-1 cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cells were lysed and immunoprecipitation and immunoblot analysis was performed with antibodies against the indicated proteins. NC, negative control.(C) RNF26 is localized to the ER and mitochondria. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Twenty-four hours after transfection, the cells were stained with Mito-Tracker Red for 15 minutes or left untreated. The cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis.D) Subcellular distribution of RNF26. The 293 cells (5×107) were infected with SeV for the indicated time points or left uninfected. Subcellular fractionation assays were performed and the cellular fractions were analyzed by immunoblots with antibodies against the indicated proteins.(E) RNF26 and MITA were colocalized to the ER. HeLa cells (2×105) were transfected with the indicated plasmids (0.5 µg each). Eighteen hours after transfection, cells were infected with SeV or HSV-1 for 6 hours or left uninfected, followed by ER-Tracker Blue/White staining for 30 minutes. Cells were fixed with 4% paraformaldehyde and subjected for confocal microscopy analysis. All experiments were repeated for at least three times with similar results.
Figure 3(A) RNF26 mediated polyubiquitination of MITA at K150. The 293 cells (5×106) were transfected with HA-Ub (1 µg) and RNF26 (1 µg) together with Flag-MITA or the indicated mutants (5 µg each). Twenty-four hours after transfection, cells were subjected IP under denatured conditions with anti-Flag and immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated.B) RNF26 targeted MITA for polyubiquitination at K150 in vitro. RNF26, MITA and its mutants were obtained by in vitro transcription and translation. Biotin-Ub, E1, UbcH5 and RNF26 were incubated with MITA or its mutants. The ubiquitination of MITA was examined by immunoblot analysis with HRP-streptavidin (top panel). The inputs of RNF26 and MITA were analyzed by immunoblots with anti-MITA and anti-RNF26 (bottom panels). All experiments were repeated for at least three times with similar results.
Figure 4(A and B) RNF26 targeted MITA for K11-linked polyubiquitination. The 293 cells (5×106) were transfected with Flag-MITA (5 µg) and RNF26 (1 µg) together with HA-Ub or its mutants (1 µg each). Twenty-four hours after transfection, cells were subjected for IP under denatured conditions with limited amount of anti-Flag (0.5 µg) so that equal amount of Flag-MITA is pulled down. The immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-Flag (lower panel). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated. Ub-AKR, all lysine residues of ubiquitin were mutated to arginine.(C) Effects of RNF26-RNAi plasmids on the expression of RNF26. In the upper panels, the 293 cells (1×106) were transfected with the expression plasmids for RNF26-Flag (0.5 µg) and HA-β-actin (0.1 µg) together with the indicated RNAi plasmids (1 µg each). Twenty-four hours after transfection, whole cell lysates were analyzed by immunoblots with anti-Flag or anti-HA. In the lower panels, 293 cells were transduced with a control or RNF26-RNAi by retrovirus mediated gene transfer. Cells (1×106) were lysed and whole cell lysates were analyzed by immunoblots with anti-RNF26 or anti-β-actin.(D) Immunoblot analysis of Flag-tagged ubiquitin expression in THP-1 cells stably transfected with Flag-Ub-K11O plasmid. Whole cell lysates of THP-1-Flag-Ub-K11O-RNF26-RNAi and control cells (1×106) were analyzed by immunoblots with antibodies against the indicated proteins. RNF26-RNAi #1 was used here and in the following experiments if not noted.(E and F) Effects of RNF26 knockdown on virus-induced K11-linked polyubiquitination of endogenous MITA. In (E), THP-1-Flag-Ub-K11O-RNF26-RNAi or control cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by IP under denatured conditions with anti-MITA. The immunoprecipitates were analyzed by immunoblots with anti-Flag (upper panels) or anti-MITA (lower panels). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated cellular or viral proteins. In (F), 293-HA-Ub-K11O cells (2×107) were transfected with a control or RNF26-RNAi plasmid (10 µg each). Twelve hours after transfection, puromycin (1 µg/mL) was added into the culture medium. The cells were selected for twenty-four hours and infected with SeV or left uninfected for the indicated time points followed by IP under denatured conditions and immunoblot analysis as in (E). All experiments were repeated for at least three times with similar results.
Figure 5A) Effects of RNF26 knockdown on virus-induced polyubiquitination of endogenous MITA. The THP-1-RNF26-RNAi or control cells (2×107) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-Ub(K48) (upper panel), anti-Ub(K63) (middle panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins.(B) Effects of RNF5 knockdown on virus-induced K11-linked polyubiquitination of endogenous MITA. The 293-HA-Ub-K11O cells (2×107) were transfected with the indicated RNAi plasmid (10 µg each). Twelve hours after transfection, the cells were selected with puromycin (1 µg/mL) for twenty-four hours and infected with SeV for the indicated time points or left uninfected. Cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated antibodies.(C) RNF26 and RNF5 competed with each other on MITA polyubiquitination. The 293 cells (5×106) were transfected with MITA (5 µg), Flag-Ub-K48O and HA-Ub-K11O (1 µg each) together with indicated amount of RNF26 and RNF5. Twenty-four hours after transfection, cell lysates were subjected to IP under denatured conditions with anti-MITA and the immunoprecipitates were analyzed by immunoblots with anti-Flag (upper panel), anti-HA (middle panel) or anti-MITA (lower panel). The whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins.(D) Effects of RNF26 knockdown on virus-triggered MITA degradation. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected. Cells were lysed and whole cell lysates were analyzed by immunoblots with antibodies against the indicated proteins (upper immunoblots). The relative protein levels of MITA in reference to β-actin were analyzed by Quantity One program and data shown are mean ± S.D. of three independent experiments (lower histographs).
Figure 6(A) Effects of RNF26 knockdown on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×105) were transfected with the IFN-β promoter reporter (0.1 µg) and the indicated RNAi plasmid (0.5 µg each). Thirty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed.(B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B)(B and C) Effects of RNF26 knockdown on virus-triggered induction of IFN-β in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (B) or ELISA analysis (C(D) Effects of RNF26 knockdown on virus-triggered induction of IFNB1 gene in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with VSV, EMCV or ECTV for the indicated time points or left uninfected before quantitative real-time PCR analysis was performed as in (B).(E and F) Effects of RNF26 knockdown on virus-triggered induction of TNFα in THP-1 cells. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected followed by quantitative real-time PCR (E) and ELISA analysis (F).(G) Effects of RNF26 knockdown on virus-triggered phosphorylation of TBK1, IRF3 and IκBα. The THP-1-RNF26-RNAi or control cells (1×106) were infected with SeV or HSV-1 for the indicated time points or left uninfected, whole cell lysates were analyzed by immunoblots with anti-p-TBK1, anti-TBK1, anti-p-IRF3, anti-IRF3, anti-p-IκBα, anti-IκBα, anti-RNF26 or anti-β-actin as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. (n = 3) of a representative experiment performed in triplicate.
Figure 7A) Effects of RNF26 knockdown on IRF3 level. In the left panel, whole cell lysates of THP-1-RNF26-RNAi or control cells (1×106) were analyzed by immunoblots with the indicated antibodies. In the right panel, cells (1×106) were subjected to quantitative real-time PCR analysis.B) Effects of RNF26 on IRF3 stability. The 293 cells (1×106) were transfected with HA-IRF3 (0.5 µg) and HA-β-actin (0.1 µg) together with RNF26 or its mutant (0.3 µg each). Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26.(C) Effects of RNF26 and its mutant on SeV-triggered activation of the IFN-β promoter. The 293 cells (2×105) were transfected with the IFN-β promoter reporter (0.1 µg) and RNF26 or its mutant (0.1 µg each). Twenty hours after transfection, cells were infected with SeV for 12 hours or left uninfected before reporter assays were performed.(D) Effects of RNF26 on IRF3 ubiquitination. The 293 cells (5×106) were transfected with Flag-IRF3 (5 µg) and RNF26 (1 µg) together with HA-Ub or its mutants (1 µg each). Eighteen hours after transfection, 3-MA (500 ng/mL) was added into culture medium for 4 hours to protect IRF3 from autophagosomal degradation during the experiment. The cell lysates were subjected to IP under denatured conditions with anti-Flag and the immunoprecipitates were analyzed by immunoblots with anti-HA (upper panels) or anti-Flag (lower panels). The whole cell lysates were analyzed by immunoblots with anti-Flag or anti-RNF26 as indicated.(E) Effects of inhibitors on RNF26-mediated destabilization of IRF3. The 293 cells (1×106) were transfected with the indicated plasmids as described in (B). Eighteen hours after transfection, 3-MA (500 ng/mL), NH4Cl (25 mM) or MG132 (100 µM) was added into culture medium for four hours. Whole cell lysates were analyzed by immunoblots with anti-HA or anti-RNF26.(F) Effects of ATG12 knockdown on RNF26-mediated destabilization of IRF3. The 293-ATG12-RNAi or control cells (1×106) were transfected with the indicated plasmids. Twenty-four hours later, whole cell lysates were analyzed by immunoblots with anti-HA, anti-RNF26 or anti-ATG12 as indicated. All experiments were repeated for at least three times with similar results. The bar graphs show mean ± S.D. (n = 3) of a representative experiment performed in triplicate.
Figure 1 (A,B) Expression of UVRAG under cell stress. HL60 and HCT116 cells were treated with doxorubicin (1 μg/ml), cisplatin (10 μm) and UV irradiation (5 min after 50 mJ/cm2) for the indicated time, and then apoptosis, autophagy and fluorescence intensity of UVRAG was assayed. Scale bar, 10 μm. (C) In parallel, the total protein extracts were used for western blot analysis.(D,E) Pharmacological inhibition (for example, 3‐methyladenine (3‐MA), 10 mM) or genetic deletion (for example, ATG5−/−) of the autophagy pathway decreases the protein and mRNA expression of UVRAG and increases apoptosis at 12 h in HL60 cells treated with doxorubicin (Doxo) and UV irradiation (n=3, *P0.05). AU, arbitrary units; C‐PARP, cleaved poly‐ADP ribose polymerase; LC3, light chain 3; mRNA, messenger RNA; PI, propidium iodide; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance‐associated gene.
Figure 2(A-C) After transfection with UVRAG shRNA or control shRNA for 48 h, HL60 (A), HCT116 (B) and HeLa (C) cells were treated with doxorubicin (Doxo, 1 μg/ml), cisplatin (Cis, 10 μm) and UV irradiation (5 min after 50 mJ/cm2) for 12 h or starvation (HBSS) for 3 h, and then apoptosis and autophagy were assayed (n=3, *P0.05).(D) Analysis of LC3 processing by autophagy in the presence or absence of lysosomal protease inhibitors pepstatin A (10 μg/ml) and E64D (10 μg/ml) after doxorubicin (1 μg/ml) treatment for 12 h. *P0.05 compared with control shRNA group.(E) After transfection with UVRAG shRNA or control shRNA for 48 h, ATG5+/+ and ATG5−/− murine embryonic fibroblasts (MEFs) were treated with doxorubicin (1 μg/ml), with or without Z‐VAD‐FMK (20 μM) for 12 h, and then apoptosis was assayed (n=3, *P0.05).(F) Nude mice were inoculated with 4 × 106 HL60 tumour cells following transfection of control (shControl) or UVRAG (shUVRAG)‐specific shRNA and treated with doxorubicin (10 mg/kg) beginning at day 7. Tumours were measured twice weekly, and volumes were calculated for 21 days (n=10, *P0.05; shControl+Doxo compared with shUVRAG+Doxo).(G) On day 21 in the experiments described in (F), apoptosis and UVRAG expression in tumour samples were assayed by TUNEL or western blotting, respectively (n=10, *P0.05). BAX, BCL2‐associated X protein; HBSS, Hank's Buffered Salt Solution; LC3, light chain 3; shRNA, short‐hairpin RNA; TUNEL, terminal deoxytransferase uridine triphosphate nick end labelling; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.
Figure 3(A) Control shRNA or UVRAG shRNA cells were transfected with BAX plasmid, and apoptosis was assayed (n=3, *P0.05).(B) In parallel, the mitochondrial membrane potential, cytochrome c release from mitochondria, and caspase‐3 and ‐9 activation in HL60 cells were analysed (n=3, *P0.05).(C) After transfection with UVRAG vector or control vector for 48 h, cells were transected or treated with BAX (1 μg), BAD (1 μg), BID (1 μg), FAS antibody (10 μg/ml) or TRAIL (100 ng/ml) for 24 h, then cell apoptosis was analysed (n=3, *P0.05).(D) Knockdown of UVRAG in BAX−/− HCT116 cells restored the sensitivity to UV treatment‐induced apoptosis (n=3, *P0.05). AU, arbitrary units; BAX, BCL2‐associated X protein; shRNA, short‐hairpin RNA; UV, ultraviolet; UVRAG, ultraviolet irradiation resistance associated gene.