Nucleophosmin is a major binding partner of SENP3. (A) Flag-tagged SENP3^C352S was expressed in human embryonic kidney (HEK)293T cells and affinity purification was performed using an anti-Flag agarose column. Bound proteins were eluted with Flag peptide, separated by SDS–polyacrylamide gel electrophoresis and analysed using mass spectrometry. (B) Left: Flag-SENP3 was expressed in HEK293T cells and immunoprecipitated (IP) with anti-Flag agarose beads. The bound material was separated by SDS–polyacrylamide gel electrophoresis and probed using western blotting with the indicated antibodies. Right: endogenous SENP3 was immunoprecipitated from HeLa cells with a rabbit polyclonal antibody and probed using western blotting with the indicated antibodies. (C) NPM1 was tested in a directed yeast two-hybrid assay for interaction with SUMO proteases as indicated. (D) HeLa cells were transfected with SV5-SENP3 and Flag-NPM1, and localization was determined by immunostaining with SV5 and Flag antibodies. Nuclei were visualized using 4,6-diamidino-2-phenylindole (DAPI) staining. MW, molecular weight; NPM1, nucleophosmin.

SENP3 catalyses the desumoylation of nucleophosmin. (A) HA-NPM1 was expressed alone or together with Flag-p14^ARF and SUMO1 or SUMO2 in the presence or absence of SV5-tagged SENP3 in human embryonic kidney (HEK)293T cells. Expression of the respective proteins was verified by immunoblotting. Western blotting with anti-tubulin shows equal loading. (B) HA-NPM1 and His-tagged versions of SUMO2 or ubiquitin were expressed in the presence of Flag-p14^ARF and SV5-SENP3 in HEK293T cells. His–SUMO2 or His–ubiquitin conjugates were recovered on Ni-NTA beads and subjected to western blotting using an HA antibody. Expression of the respective proteins was monitored by western blotting of cell lysates. Asterisks indicate a crossreactivity of the HA antibody with overexpressed SV5-tagged SENP3. (C) HEK293T cells were depleted from SENP3 or SENP5 by siRNA duplexes and transfected with the indicated plasmids. His–SUMO2 conjugates were recovered on Ni-NTA beads and subjected to western blotting using an HA antibody. Expression of the respective proteins was monitored by western blotting of cell lysates. (D) ³⁵S-labelled NPM1, generated by in vitro transcription/translation, was first incubated with recombinant E1 enzyme, E2 enzyme and either SUMO1 (lanes 2–6) or SUMO2 (lanes 7–11) in the presence of ATP. In the control reaction, SUMO was omitted (lane 1). Following the modification reaction, SENP3 (lanes 3,8) and SENP5 (lanes 5,10) or the catalytically inactive mutants SENP3^C532S (lanes 4,9) and SENP5^C713S (lanes 6,11), generated by in vitro translation/transcription, were added. The amounts of SENP3, SENP5 and the respective catalytically inactive mutants are shown below. HA, haemagglutinin; NPM1, nucleophosmin; siRNA, short interfering RNA.

Downregulation of SENP3 interferes with pre-ribosomal RNA processing. (A) Diagram summarizing the main steps of rRNA processing. (B,C) HeLa cells were transfected with siRNA duplexes targeting NPM1, SENP5 or SENP3, as indicated. Transfection of a scrambled oligonucleotide served as a control. At 72 h after transfection, cells were pulse labelled with ³²P-orthophosphate for 1 h and chased for 2 h. An equal amount of RNA was loaded into each lane. Ethidium bromide (EtBr) staining of 28S and 18S rRNA is shown. Downregulation of the respective proteins was analysed by western blotting. (D) The signal intensities of the 28S and 32S rRNA forms were quantified by phosphoimager analysis and the 28S:32S ratio was calculated. ETS, external transcribed spacer; ITS, internal transcribed spacer; NPM1, nucleophosmin; siRNA, short interfering RNA.

Constitutive SUMO2 conjugation of NPM1 interferes with pre-ribosomal RNA processing. (A) HeLa cells expressing Flag-NPM1 or Flag-NPM1–SUMO2 from a tetracycline-inducible promoter were transfected with an siRNA duplex targeting NPM1 or a control siRNA. At 72 h after transfection, in vivo labelling and processing of the samples was carried out as described in Fig 3. (B) The signal intensities were quantified as described in Fig 3D. (C) Homogenous expression and localization of Flag-NPM1 or Flag-NPM1–SUMO2 was monitored by indirect immunofluorescence. EtBr, ethidium bromide; NPM1, nucleophosmin; siRNA, short interfering RNA.