Fig 1: The SUMO E3-ligase domain of RanBP2 is required for the repression of IL6 in HAP1 and HEK293 cells.The SUMO E3-ligase domain of RanBP2 was targeted by CRISPR/Cas9 in HAP1 cells (A-E) and HEK293 cells (F-L). (A) A schematic diagram of the region of the RanBP2 gene in HAP1 cells targeted by CRISPR/Cas9 loaded with the guide RNA “gRNA-dE3-1#”. Note that the insertion “ins” in the RanBP2-E3ins cell line is indicated. (B) cDNA amplification, using p2F and p2R primers indicated in (C), of RanBP2 mRNA. Note that the reaction with the RanBP2-E3ins cell lysates produced an amplicon which was smaller than from unmodified HAP1 cells (“WT HAP1”). (C) Schematic of the modified region in the RanBP2 mRNA. Also indicated are the PCR primers “p2F”and “p2R” for cDNA amplification. Note that the mRNA produced from the modified RanBP2-E3ins gene lacks exon 21. (D-E) Unmodified and RanBP2-E3ins HAP1 cells were transfected with an intron-containing version of IL6-HA (IL6-1i-HA) and histone 1B-GFP (H1B-GFP). Cell lysates were collected 24 h post-transfection, and IL6-HA was immunoprecipitated with mouse anti-HA antibody and protein G beads (Sigma), separated by SDS-PAGE and immunoblotted with a rabbit anti-HA antibody (top panel). For the detection of other proteins, cell lysates were directly separated by SDS-PAGE and immunoblotted with antibodies against GFP, RanBP2, RanGAP1 and a-tubulin. Note that RanBP2-E3ins cells lacked sumoylated RanGAP1 but expressed RanBP2 at similar levels to unmodified HAP cells (D). IL6-HA and H1B-GFP protein levels were quantified using densitometry analysis and the ratio of IL6-HA/H1B-GFP was normalized to unmodified HAP1 cells, with each bar representing the average of three independent experiments ± SEM (E). (F) A schematic diagram of the region of the RanBP2 gene in HEK293 cells targeted by CRISPR/Cas9 loaded with the guide RNA “gRNA-dE3-1#” to generate the RanBP2-dE3-1 clone or with “gRNA-dE3-3#” to generate the RanBP2-dE3-2 clone. Also indicated are the PCR amplification primers “p1F” and “p1R”, and the regions inserted “f1-ins” and “f2-ins” or regions deleted “f1-del” and “f2-del” in each RanBP2 allele present in the “RanBP2-dE3” clones. (G-I) cDNA analysis similar to (B-C), except for RanBP2-dE3-1 in (G-H) and for RanBP2-dE3-2 in (I-J). (K-L) As in (D-E), except that unmodified, RanBP2-dE3-1 and RanBP2-dE3-2 HEK293 cells were transfected, and cell lysates were directly analyzed by SDS-PAGE and immunoblotting. Note that RanBP2-dE3-1 and RanBP2-dE3-2 HEK293 cells were not assessed in parallel, but each compared to their own parental unmodified cells. The ratio of IL6-HA/HIB-GFP in each RanBP2-dE3 cell line were normalized to unmodified parental HEK293 cells, with each bar representing the average of two independent experiments ± SEM. *P = 0.01–0.05 (Student’s t-test).
Fig 2: RanBP2 suppresses the translation of IL6 mRNA.(A) U2OS cells were infected with lentivirus containing shRNA1 or shRNA3 directed against RanBP2, or scrambled shRNA (“control shRNA”). Four days post-infection, cell lysates were collected, separated by SDS-PAGE, and immunoblotted for nucleoporins using mAb414, which recognizes RanBP2, and a-tubulin as a loading control. (B-C) U2OS cells were infected with lentivirus that delivered shRNA1 or shRNA3 against RanBP2 or control virus. Three days post-infection, cells were transfected with plasmids containing either the IL6-?i-HA, insulin-?i-HA, or ß-globin-i-HA genes. 18–24 h post-transfection cell lysates were collected and separated by SDS-PAGE. The level of each protein was analyzed by immunoblot for HA, and a-tubulin as a loading control (B). The levels of each HA-tagged protein and a-tubulin were quantified using densitometry analysis. The HA/tubulin ratio was normalized to control shRNA-treated cells and plotted, with each bar representing the average of three independent experiments ± SEM (C). (D) As in (B) except that cell lysates (left panel) or supernatant precipitated by TCA (right panel) were collected, separated by SDS-PAGE and immunoblotted with antibodies against HA and a-tubulin. (E-F) As in (B) except that RNA was purified from cell lysates and separated on a denaturing agarose gel. The levels of IL6-HA mRNA and a-tubulin were assessed by northern blot, while the ribosomal RNA was detected by ethidium bromide (E). IL6-HA and a-tubulin mRNA levels were quantified using densitometry analysis. The IL6-HA/tubulin ratio was normalized to control shRNA-treated cells and plotted with each bar representing the average of three independent experiments ± SEM (F). (G-H) Control and RanBP2-depleted cells were transfected with an intronless version of IL6-HA (IL6-?i-HA) plasmid for 14–18 hr, then fixed, permeabilized, and stained for mRNA using a fluorescent in situ hybridization (FISH) probe directed against IL6. The cells were imaged (G) and total integrated fluorescence was assessed in the cytoplasm and nucleus (H). For each experiment at least 20 cells were assessed with each bar representing the average of three independent experiments ± SEM. Scale bar = 10 µm. *P = 0.01–0.05, **P = 0.001–0.01, ***P < 0.001, n.s. indicates no significant difference (Student’s t-test).
Fig 3: Human adenovirus interacts with RANBP2 for nuclear import and viral assembly.(A) DAPI nuclear (blue) and RANBP2 (green) staining of NC-siRNA and RANBP2-siRNA treated HEK293 cells, infected with HAdV-D37 at an MOI of 0.1 for 24 and 48 hrs, respectively. Scale bar = 10 µm. (B) ImageJ quantification of DAPI fluorescence per cell, normalized to uninfected control cells (n = 100) and plotted as the percent of uninfected control fluorescence. (C) Transmission electron microscopy of NC-siRNA and (D) USP9x siRNA, and (E) RANBP2-siRNA treated HEK293 cells infected with HAdV-D37 at an MOI of 0.1 for 72 hrs. The specific MOI and time-points were chosen to establish viral replication while minimizing early cell death; HAdV-D37 infection at an MOI of =1 in HEK293 cells leads to significant cytopathic effect within 24 hpi. White arrows over the nuclei show large paracrystalline viral arrays. Higher magnifications are shown for NC-siRNA (F) and RANBP2-siRNA (G) treated cells. The final data in (B) are presented as the mean ± SD of triplicate experiments. Statistical significance was performed with unpaired t-test (two-tailed). No statistically significant differences were seen.
Fig 4: Schematic overview of pIIIa, USP9x, and RANBP2 interactions.During infection, viral pIIIa bound to host USP9x. MG132 proteasome inhibitor treatment did not stabilize pIIIa or RANBP2 steady state concentrations. On the other hand, viral replication increases in USP9x knockout (KO) or knockdown (KD) cells suggesting that USP9x favors the host during infection by negatively regulating viral replication. Viral pIIIa binds to nucleoporin RANBP2, using its nuclear import function for translocation to the nucleus. RANBP2 knockdown does not impact the early stages of infection–there is no change in nuclear DNA entry or nuclear chromatin condensation. However, RANBP2 knockdown reduces viral replication, leading to accumulation of defective assembly products in the infected cells. CRM1 modulates RANBP2-pIIIa interactions and cytoplasmic transport. USP9x and RANBP2 bind to different sites of C-terminus pIIIa. These interactions are important across different HAdV species.
Fig 5: RanBP2 SUMO E3-ligase activity is not required for the stability of AGO1 in HAP1 and HEK293 cell lines.(A) Unmodified and RanBP2-E3ins HAP1 cells were lysed, separated by SDS-PAGE, and immunoblotted with antibodies against AGO1, AGO2, RanBP2, RanGAP1, and a-tubulin. (B) Unmodified HAP1 and RanBP2-E3ins cells were treated with cycloheximide (CHX, 100 µM) for various amounts of time to block further translation and with or without proteasome inhibitor MG132 (50 µM) (MG132 “+” or “-”). Cell lysates were collected, separated by SDS-PAGE, and immunoblotted with antibodies against AGO1, a-tubulin, RanGAP1 and RanBP2. (C) As in (A), except for unmodified and RanBP2-dE3-2 HEK293 cells.
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