Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Immunofluorescence images and quantification (n = 20) of SUMO1 (green) and SUMO2/3 (red) conjugation in RAW264.7 cells with or without STM 14028S infection (4 hpi). Scale bar, 10 µm. b Core SUMO cycle enzymes and their corresponding primary genes (blue). c, d Transcriptomic and proteomic analyses of SUMO cycle enzyme expression in RAW264.7 cells infected with STM 14028S vs uninfected controls (4 hpi; n = 3). e UBC9 mRNA expression in RAW264.7 cells during STM 14028S infection (0-6 hpi; n = 3). f Immunoblot analysis and quantification of UBC9 protein levels in STM 14028S-infected RAW264.7 cells (0-6 hpi; n = 3). *P < 0.05; ***P < 0.001; ns, not significant.

Article Snippet: Unique primary antibodies used in this study included UBC9 (CST, #4786), SUMO1 (Proteintech, 67557-1-lg), SUMO2/3 (Proteintech, 67154-1-lg), arginine-GlcNAcylation antibody (Abcam, EPR18251), Myd88 (Proteintech, 67969-1-lg), HSPA8 (Proteintech, 10654-1-AP), PDCD4 (Proteintech, 84162-3-RR).

Techniques: Immunofluorescence, Conjugation Assay, Infection, Expressing, Western Blot

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Immunofluorescence images and quantification (n = 20) of SUMO1 (green) and SUMO2/3 (red) conjugation in RAW264.7 cells infected with STM 14028S over a 0-6 hrs time course. Scale bar, 10 µm. b Immunofluorescence images and quantification (n = 20) of SUMO1 (green) and SUMO2/3 (red) conjugation in RAW264.7 cells infected with STM WT or Δ ssaV or left uninfected (4 hpi). Scale bar, 10 µm. c Schematic of the high-content screening (HCS) workflow used to identify T3SS-2 effector(s) required for subversion of host SUMOylation. d SUMO2/3 suppression rates in RAW264.7 cells infected with STM WT or T3SS-2 effector knockout strains (4 hpi). For each sample, fluorescence intensity (FI) was measured across three fields (20 cells per field) to calculate the mean FI (MFI). Suppression rate = (MFI_uninfected - FI_test) / (MFI_uninfected - MFI_WT). e Representative immunofluorescence images of SUMO2/3 conjugation in RAW264.7 cells infected with STM WT, Δ ssaV , Δ sseK1 , or left uninfected (4 hpi). f Immunoblot analysis of SUMO2/3 conjugates in RAW264.7 cells infected with STM WT or Δ sseK1 or left uninfected (4 hpi). *P < 0.05; ***P < 0.001; ns, not significant.

Article Snippet: Unique primary antibodies used in this study included UBC9 (CST, #4786), SUMO1 (Proteintech, 67557-1-lg), SUMO2/3 (Proteintech, 67154-1-lg), arginine-GlcNAcylation antibody (Abcam, EPR18251), Myd88 (Proteintech, 67969-1-lg), HSPA8 (Proteintech, 10654-1-AP), PDCD4 (Proteintech, 84162-3-RR).

Techniques: Immunofluorescence, Conjugation Assay, Infection, High Content Screening, Knock-Out, Fluorescence, Western Blot

Journal: bioRxiv

Article Title: A bacterial effector blocks SUMOylation by steric occlusion of UBC9 via arginine-GlcNAcylation

doi: 10.64898/2026.03.06.710069

Figure Lengend Snippet: a Crystal structure of the UBC9-SUMO1 complex (PDB: 2UYZ). b Predicted structure of the UBC9-SUMO2 complex generated using AlphaFold3. Enlarged views of the UBC9-SUMO1 and UBC9-SUMO2 interfaces are highlighted in black boxes. Residues forming hydrogen bonds with UBC9 R17 are shown in stick representation. c Purification of 6×His-UBC9 from E. coli BL21(DE3) co-expressing pSseK1 and pUBC9, followed by immunoblot analysis of UBC9 Arg-GlcNAcylation. d Purification of SUMO1 and SUMO2 proteins from E. coli BL21(DE3) harboring pSUMO1 or pSUMO2, respectively. M, molecular weight marker. e, f MST analysis of the binding affinities between SUMO1 or SUMO2 and unmodified UBC9 or Arg-GlcNAcylated UBC9.

Article Snippet: Unique primary antibodies used in this study included UBC9 (CST, #4786), SUMO1 (Proteintech, 67557-1-lg), SUMO2/3 (Proteintech, 67154-1-lg), arginine-GlcNAcylation antibody (Abcam, EPR18251), Myd88 (Proteintech, 67969-1-lg), HSPA8 (Proteintech, 10654-1-AP), PDCD4 (Proteintech, 84162-3-RR).

Techniques: Generated, Purification, Expressing, Western Blot, Molecular Weight, Marker, Binding Assay

Journal: bioRxiv

Article Title: Human mascRNA interacts with SUMO E1 and reshapes nuclear SUMOylation

doi: 10.64898/2026.01.19.700226

Figure Lengend Snippet: Purity and activity of recombinant SUMO E1 (SAE1/2) used for in vitro assays of association with synthetic mascRNA. ( A ). SDS-PAGE and Coomassie stain of purified Hist-tagged SAE1/2 complex (Genscript). ( B ). In vitro SUMOylation assay of purified SAE1/2 complex using RANGAP1 as a substrate, and SUMO1 immunoblot of assay products. ( C ). In vitro auto-SUMOylation of SAE2 by incubation of the SAE1/2 complex with SUMO1 and ATP, and SDS-PAGE analysis as indicated. ( D ). EMSA assays using SAE1/2 complex from two sources (N-terminal His-tag by Genscript, non-His-tagged from R&D) and mascRNA, as indicated. Heparin was used as indicated as non-specific polyanion competitor in some reactions. mascRNA-SAE1/2 complexes are highlighted. An additional higher MW complex was detected only with the R&D SAE1/2 preparation. The positions of the gel wells is indicated. ( E ). EMSA using SAE1/2 and mascRNA, in the presence of increasing amounts of yeast total RNA. Mass of yeast total RNA relative to mascRNA is indicated. ( F ). SAE1 and SAE2 immunoblots of lysates and IP eluates from SAE2 CLIP experiments, related to main .

Article Snippet: Chambers were stained with SAE2 (Bethyl A302-925A), or SUMO1 (Santa Cruz sc-5308) primary antibodies 1/1000, overnight at +4 °C, on slow motion (∼10 rpm) inside a humidified box, protected from light.

Techniques: Activity Assay, Recombinant, In Vitro, SDS Page, Staining, Purification, Western Blot, Incubation

Journal: bioRxiv

Article Title: Human mascRNA interacts with SUMO E1 and reshapes nuclear SUMOylation

doi: 10.64898/2026.01.19.700226

Figure Lengend Snippet: SAE1/2 in vitro activity and assembly in the presence of mascRNA. ( A ). In vitro SUMOylation assay of purified SAE1/2 complex using RANGAP1 as a substrate in the presence of mascRNA as indicated, and SUMO1 immunoblot of assay products. ( B ). Native gel analysis of the SAE1/2 complex in the absence and presence of increasing amounts of mascRNA. SAE1/2 complex as one main band, and no dissociation of the two SAE subunits in the presence of mascRNA was observed. ( C ). In vitro auto-SUMOylation of SAE2 by incubation of the SAE1/2 complex with SUMO1 and ATP, in the absence or presence of mascRNA, and SDS-PAGE analysis as indicated. No inhibition of the SAE1/2 enzymatic activity was observed.

Article Snippet: Chambers were stained with SAE2 (Bethyl A302-925A), or SUMO1 (Santa Cruz sc-5308) primary antibodies 1/1000, overnight at +4 °C, on slow motion (∼10 rpm) inside a humidified box, protected from light.

Techniques: In Vitro, Activity Assay, Purification, Western Blot, Incubation, SDS Page, Inhibition

Journal: bioRxiv

Article Title: Human mascRNA interacts with SUMO E1 and reshapes nuclear SUMOylation

doi: 10.64898/2026.01.19.700226

Figure Lengend Snippet: SAE2 nuclear localization and its modulation by mascRNA (A). SYPRO Ruby stain of urea-eluted proteins from NRS and SAE2-IPs. ( B ). SAE2-IP proteomics vs NRS control in untreated cells. Heatmap of unique peptide number per identified protein per IP and subcellular localization (Uniprot). ( C ). Cellular fractionation and mascRNA northern blot from nuclear and cytoplasmic fractions; U6 was probed as a control of the fractionation. ( D ). SAE2 and SUMO1 immunofluorescence of untreated cells. Scale bar: top row 2 μm, bottom 3 μm. ( E ). mascRNA and scrambled oligo treatment of 293FT cells, SAE2 and SUMO1 immunofluorescence. Scale bar: 20 μm. ( F ). Quantifications and statistic test of surface area of SAE2 + signal and SAE2 + particle numbers after scrambled oligo control (scr) and mascRNA treatments. Data are shown as means ± S.D; Student’s t -test ** p =0.0025, **** p <0.0001, SUMO1: n.s, p =0.1493, n =7 FOV per condition.

Article Snippet: Chambers were stained with SAE2 (Bethyl A302-925A), or SUMO1 (Santa Cruz sc-5308) primary antibodies 1/1000, overnight at +4 °C, on slow motion (∼10 rpm) inside a humidified box, protected from light.

Techniques: Staining, Control, Cell Fractionation, Northern Blot, Fractionation, Immunofluorescence

Journal: bioRxiv

Article Title: Human mascRNA interacts with SUMO E1 and reshapes nuclear SUMOylation

doi: 10.64898/2026.01.19.700226

Figure Lengend Snippet: Detection of protein SUMOylation and its modulation by mascRNA transfection. ( A ). PBRM1 IP and immunoblot, related to main . ( B ). Transient FLAG-SUMO2 and FLAG-SUMO3 expression for the tagging and FLAG immunocapture of SUMOylated proteins. Immunoblot with anti-SUMO2/3 Ab. ( C ). Transient FLAG-SUMO2 and FLAG-SUMO3 expression for the tagging and FLAG immunocapture of SUMOylated proteins. Immunoblot with anti-FLAG Ab (M2). ( D ). The same lysate and IP samples as shown in panels (B) and (C) were immunoblotted with anti-SMARCA4 (BRG1) Ab. Poor SMARCA4 presence in the IP eluates and no higher MW signal indicative of SUMOylation were observed. ( E ). Same as in D, immunoblot for ARID2. Poor ARID2 presence in the IP eluates and no higher MW signal indicative of SUMOylation were observed. ( F ), ( G ). MascRNA transfection induces reduced nuclear protein SUMOylation. Replicate experiments related to main . Scrambled oligo (scr) was used as a control, cell fractionation as in main . SUMO1 conjugates are highlighted. Histone3 was used as control of cell fractionation. WCL: whole cell lysate, Nuc: nuclear fraction, Cyto: cytoplasmic fraction. Quantifications are shown in main . ( H ). STRING network analysis of proteins enriched in SUMO1 IP, from cells transfected with mascRNA or scrambled control oligo. Select statistically enriched Gene Ontology terms (FDR< 0.05) are shown; the proteins are colored by term enrichment as indicated.

Article Snippet: Chambers were stained with SAE2 (Bethyl A302-925A), or SUMO1 (Santa Cruz sc-5308) primary antibodies 1/1000, overnight at +4 °C, on slow motion (∼10 rpm) inside a humidified box, protected from light.

Techniques: Transfection, Western Blot, Expressing, Control, Cell Fractionation

Journal: bioRxiv

Article Title: Human mascRNA interacts with SUMO E1 and reshapes nuclear SUMOylation

doi: 10.64898/2026.01.19.700226

Figure Lengend Snippet: ( A ). Transient FLAG-SUMO1 expression for the tagging and immunocapture of SUMOylated proteins. ( B ). Immunoblot of SUMOylated KAP1 protein from FLAG-SUMO1 immunoprecipitates. ( C ). Immunoblot of SUMOylated PBRM1 protein from FLAG-SUMO1 immunoprecipitates. ( D ). Inhibition of SUMOylation in cell culture by TAK981. Cellular fractions were blotted for SUMO1. Histone3 and alpha-tubulin were used as controls of cell fractionation. WCL: whole cell lysate, Nuc: nuclear fraction, Cyto: cytoplasmic fraction. ( E ). MascRNA transfection induces reduced nuclear protein SUMOylation. Scrambled oligo (scr) was used as a control, cell fractionation as in (D). SUMO1 conjugates are highlighted. One representative replicate experiment shown, see also and G. ( F ). Quantification of SUMO1 conjugates (area highlighted in (E)) from three replicate mascRNA vs scrambled control oligo experiments. Data are shown mean ± S.D of densitometric signal at MW > 80 kDa, Student’s t -test, * p =0.040, error bars: SD.

Article Snippet: Chambers were stained with SAE2 (Bethyl A302-925A), or SUMO1 (Santa Cruz sc-5308) primary antibodies 1/1000, overnight at +4 °C, on slow motion (∼10 rpm) inside a humidified box, protected from light.

Techniques: Expressing, Western Blot, Inhibition, Cell Culture, Cell Fractionation, Transfection, Control

Journal: bioRxiv

Article Title: Human mascRNA interacts with SUMO E1 and reshapes nuclear SUMOylation

doi: 10.64898/2026.01.19.700226

Figure Lengend Snippet: ( A ). SDS-PAGE analysis of Urea and SUMO1-peptide eluates of SUMO1 IP. IgG IP was used as a control. ( B ). SUMO1 immunoblot of lysates and urea eluates of SUMO1 IPs, three replicates per condition. SUMO1 conjugates are highlighted. ( C ) SUMOylated KAP1 detection in SUMO1 IPs, as a verification of the specificity of the approach. Below: table with number of KAP1 unique peptides identified in IgG and SUMO1 IPs. ( D ) Numbers of unique and total peptides identified in IP eluates of IgG and SUMO1 IP replicates. ( E ). Pie chart of proteins enriched in SUMO1 IP and how their peptide counts changed in cells treated with mascRNA vs scrambled control oligo, and binomial statistic test of decreased vs increased groups. ( F ). Volcano plot for the proteins identified in the SUMO1 IP, mascRNA vs control oligo. ( G ). STRING network analysis of the proteins that showed decreased peptide counts in the SUMO1 IP, mascRNA vs control oligo. Select statistically Gene Ontology enriched terms (FDR< 0.05) are shown; proteins are colored by term enrichment as indicated. ( H ) SUMO1 and PML immunofluorescence for co-localization in cells treated with 70 nM TAK981 SUMO E1 inhibitor, DMSO was used as a control. Scale bar, 10 μm. ( I ). Image analysis for PML positive (PML+) particles. Up: counts of PML+ particles in the two conditions. Student’s t- test ** p <0.01, mean and ± S.D. Down: pixel-by-pixel Pearson’s Correlation Coefficient of PML and SUMO1 signal, Student’s t- test **** p <0.0001, mean and ± S.D., n = 6 FOVs per condition. ( J ) SUMO1 and PML immunofluorescence for co-localization in cells treated with 10 nM mascRNA, scrambled oligo was use as a control. Scale bar, 10 μm. ( K ). Image analysis for PML positive (PML+) particles. Up: counts of PML+ particles in the two conditions. Student’s t- test ** p <0.01, mean and ± S.D. Down: pixel-by-pixel Pearson’s Correlation Coefficient of PML and SUMO1 signal, Student’s t- test ** p <0.01, mean and ± S.D., n =39 FOV 5’P-scr vs n =38 FOV 5’P-masc.

Article Snippet: Chambers were stained with SAE2 (Bethyl A302-925A), or SUMO1 (Santa Cruz sc-5308) primary antibodies 1/1000, overnight at +4 °C, on slow motion (∼10 rpm) inside a humidified box, protected from light.

Techniques: SDS Page, Control, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Human mascRNA interacts with SUMO E1 and reshapes nuclear SUMOylation

doi: 10.64898/2026.01.19.700226

Figure Lengend Snippet: ( A ) ZNF106 immunoblot of lysates and urea eluates of SUMO1 IPs, three replicates per condition. SUMO1 conjugates are highlighted in red; below: number of unique ZNF106 peptides identified in SUMO1 IP proteomics. ( B ) BEND3 immunoblot of lysates and urea eluates of SUMO1 IPs, three replicates per condition. SUMO1 conjugates are highlighted in red; below: number of unique BEND3 peptides identified in SUMO1 IP proteomics. ( C ) Quantification of SUMO1 conjugates from panel (B). **p=0.0021, Student’s t test, n =3, error bars: SD. ( D ) Detection of SUMO1-modified BEND3 in FLAG IPs, with lysates from cells transiently expressing the FLAG constructs indicated. ( E ) Detection of SUMO1-modified ZNF106 in FLAG IPs, with lysates from cells transiently expressing the FLAG constructs indicated. ( F ) Effect of mascRNA vs scrambled oligo transfection in cells expressing FLAG-SUMO1; SUMO1 immunoblot of lysates and FLAG-IP eluates. FLAG-SUMO1 conjugates are highlighted in red. ( G ) ZNF106 immunoblot in lysates and FLAG IP eluates of FLAG-SUMO1 expressing cells, transfected with mascRNA and scrambled oligo, two replicate experiments. FLAG-SUMO1 conjugates are highlighted in red. ( H ) Quantification of SUMO1 conjugates from panel (G), normalized to scrambled oligo signal. Error bars: min-max, n =2.

Article Snippet: Chambers were stained with SAE2 (Bethyl A302-925A), or SUMO1 (Santa Cruz sc-5308) primary antibodies 1/1000, overnight at +4 °C, on slow motion (∼10 rpm) inside a humidified box, protected from light.

Techniques: Western Blot, Modification, Expressing, Construct, Transfection