SENP3 and SENP5 codepletion cause nucleolar SUMO protein accumulation. (A) HeLa cells were transfected with siRNAs directed against SENP3 and SENP5, either singly or in combination. 72 h after transfection, depletion was confirmed by Western blotting. (B) Cells depleted of SENP3, SENP5, or both proteins were stained with antibodies against B23/nucleophosmin (B23; red) and either SUMO-1 (left; green) or SUMO-2/3 (right; green).

B23/nucleophosmin regulates SENP3 and SENP5 abundance. (A) HeLa cells were transfected with control oligonucleotides (left lane), siRNAs for codepletion of SENP1 and SENP2 (second lane), siRNAs for codepletion of SENP3 and SENP5 (third lane), or siRNAs for depletion of B23/nucleophosmin (right lane). After 72 h, the cells were harvested and immunoblotted with the indicated antibodies, where actin serves as a loading control. (B) U2OS cells stably expressing either GFP-SENP3 (top) or GFP-SENP5 (bottom) were transfected with control or B23/nucleophosmin siRNA, as indicated. After 72 h, the cells were washed and fixed with 4% paraformaldehyde and stained with antibodies against B23/nucleophosmin (red). Signals from the GFP-SENP3 and GFP-SENP5 fusion proteins are shown in green. (C) As in A, HeLa cells were transfected with control oligonucleotides or with siRNAs for depletion of B23/nucleophosmin. After 72 h, the cells were stained with antibodies against B23/nucleophosmin (red) and against either SUMO-1 or SUMO-2/3 (green), as indicated. (D) 48 h after transfection with control or B23/nucleophosmin siRNAs, HeLa cells were treated for 6 h with 20 μM of proteasome inhibitor MG132 (+) or DMSO (−). The cells were harvested and subjected to Western blotting analysis with antibodies against SENP3, SENP5, B23/nucleophosmin, and actin, as indicated.

SENP3 and SENP5 colocalize and associate with B23/nucleophosmin. (A) U2OS-derived cell lines expressing GFP-SENP3 and GFP-SENP5 were transfected with a plasmid for expression of dsRed-fibrillarin (top). After 48 h, the cells were fixed and immunostained with monoclonal anti-UBF antibodies. The left column shows GFP, dsRed, and anti-UBF in the context of the nucleus, with DNA stained in blue using Hoechst 33342. The same cells were transfected with dsRed-B23/nucleophosmin (bottom). (B) Rabbit IgG (IgG), anti-xSENP3, or anti-xB23/nucleophosmin (xB23/NPM) antibodies were used for immunoprecipitation from 100 μl of interphase XEE (top). The samples were subjected to SDS-PAGE and immunoblotting with anti-xSENP3 or anti-B23/nucleophosmin antibodies. To test xSENP5 binding to xB23/nucleophosmin, 25 μl of XEE containing in vitro–translated FLAG-tagged xSENP5 was mixed with equal volume of interphase XEE. Immunoprecipitations were performed with anti-FLAG or anti-xB23/NPM antibodies (bottom). The samples were subjected to immunoblotting with anti-FLAG or anti-xB23/NPM antibodies. (C) Anti-xSENP3 was used for immunoprecipitation from IgG-treated control (+) or xB23/NPM-depleted (−) XEEs. The samples were subjected to SDS-PAGE and Western blotting with anti-xSENP3, -Rpl26, -RplP0, or -xB23/NPM antibodies, as indicated on the right.

Depletion of SENP3 or SENP5 cause defects in ribosome biogenesis. (A) HeLa cells were transfected with control oligonucleotides (Control) or siRNAs directed against SENP3 (ΔSENP3) and SENP5 (ΔSENP5) or both SENP3 and SENP5 (ΔSENP3/5). 72 h later, the cells were pulsed with [³H]uridine for 25 min, followed by washing and incubation with fresh media supplemented with 1 mg/ml of cold uridine for the indicated times (in hours). At each time point, equal numbers of cells were harvested for analysis of rRNA synthesis and processing. (Bottom) Western blots of cell extracts to monitor SENP3 and SENP5 and Western blotting of actin as a loading control. Ethidium bromide staining shows the amount of total RNA loaded in each lane. (B) The amounts of 28S and 32S RNAs were quantitated within three independent experiments as in A after 2 h. Ratios of 32S to 28S RNAs were calculated for each experiment. Bars represent the mean values calculated from the three experiments ± SD. (C) The amount of 47S pre-rRNA transcript was quantitated at the initiation of the chase period (0 h) within three independent experiments as in A and normalized to the levels found in cells treated with control oligonucleotides. Bars represent the mean of normalized values from the three experiments ± SD.

Enhanced SUMO modification of RPL37A and GNL2 after depletion of SENP3 and SENP5. U2OS stably expressing full-length His-tagged versions of SUMO-1 and SUMO-2 (His-SUMO1F and His-SUMO2F) were transfected with siRNAs for codepletion of SENP3 and SENP5 or with control oligonucleotides. After 72 h, the cells were harvested and His₆-SUMO–modified proteins were isolated. Total cell extracts and His₆ affinity fractions were analyzed by Western blotting with antibodies against RPL37A (top), GNL2 (middle), or B23/nucleophosmin (bottom).