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First published online 10 February 2009
doi: 10.1242/jcs.044461

Journal of Cell Science 122, 678-686 (2009)
Published by The Company of Biologists 2009
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Nucleolar structure and function are regulated by the deubiquitylating enzyme USP36

Akinori Endo1, Masaki Matsumoto2, Toshifumi Inada3, Akitsugu Yamamoto4, Keiichi I. Nakayama2, Naomi Kitamura1 and Masayuki Komada1,*

1 Department of Biological Sciences, Tokyo Institute of Technology, Yokohama 226-8501, Japan
2 Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan
3 Department of Molecular Biology, Nagoya University, Nagoya 464-8602, Japan
4 Department of Bio-science, Nagahama Institute of Bio-science and Technology, Nagahama 526-0829, Japan


Figure 1
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  		Fig. 1. USP36 localizes to nucleoli. (A-A'') HeLa cells were transfected with Flag-USP36, and double-stained with anti-Flag (A) and anti-nucleolus (A', red) antibodies. In A', nuclei were also stained in blue. A'' is the merged image. Arrowheads indicate typical nucleoli. Asterisks indicate untransfected cells. Scale bar: 10 µm. (B) HeLa cell lysate immunoblotted with pre-immune serum or anti-USP36 antiserum. Arrowhead indicates full-length USP36. (C) Lysates of HeLa cells transfected with or without Flag-USP36 immunoblotted with anti-USP36 and anti-{alpha}-tubulin antibodies. (D-D'') HeLa cells double-stained with anti-USP36 (D) and anti-nucleolus (D', red) antibodies. D'' is the merged image. Arrowheads indicate typical nucleoli. Scale bar: 10 µm. (E) Immuno-EM analysis of HeLa cells with anti-USP36 antibody. Nucleoli are indicated by arrowheads. N and C indicate the nucleoplasm and cytoplasm, respectively. Scale bar: 5 µm.

 

Figure 2
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  		Fig. 2. The C-terminal region targets USP36 to nucleoli. (A) Schematic structure of human USP36. Five basic stretches in the C-terminal region, as well as the Cys and His boxes, are indicated. Regions covered by the four truncated mutants used in this study are also shown. (B) Lysates of HeLa cells transfected with Flag-tagged wild-type and truncated USP36 constructs were immunoblotted with anti-Flag antibody. Open arrowheads and asterisk indicate the USP36 constructs and a non-specific band, respectively. (C-F'') HeLa cells were transfected with Flag-tagged USP361-420 (C-C''), USP36421-800 (D-D''), USP36801-1121 (E-E'') or USP36{Delta}801-1121 (F-F''), and double-stained with anti-Flag (C-F) and anti-nucleolus (C'-F') antibodies. C''-F'' are merged images in which nuclei were also stained in blue. Arrowheads in E-E'' indicate typical nucleoli. Scale bars: 10 µm.

 

Figure 3
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  		Fig. 3. USP36 exhibits DUB activity in nucleoli. (A) COS-7 cells were transfected with HA-tagged wild-type USP36 or USP36C131A together with Flag-ubiquitin for 2 days, and treated with MG132 for 8 hours. Their lysates were immunoblotted with anti-Flag, anti-HA and anti-{alpha}-tubulin antibodies. (B-C'') HeLa cells transfected with Flag-tagged wild-type USP36 (B-B'') or USP36C131A (C-C'') and double-stained with anti-Flag (B,C) and FK2 (B',C') antibodies. B'' and C'' are merged images. Arrowheads indicate typical nucleoli. Asterisks indicate untransfected cells. Scale bars: 10 µm.

 

Figure 4
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  		Fig. 4. USP36 deubiquitylates and stabilizes NPM. (A) COS-7 cells transfected with HA-tagged wild-type USP36 or USP36C131A with Flag-ubiquitin for 2 days, and treated with MG132 for 8 hours. Lysates were immunoprecipitated with anti-NPM antibody and immunoblotted with anti-Flag (top) and anti-NPM (second from top) antibodies. Expression levels of HA-tagged USP36 constructs and endogenous {alpha}-tubulin (loading control) assessed by immunoblotting of the total cell lysates with anti-HA (third from top) and anti-{alpha}-tubulin (bottom) antibodies. Asterisk indicates the smear of polyubiquitylated NPM. (B) Lysates of COS-7 cells transfected with HA-tagged wild-type USP36 or USP36C131A immunoprecipitated with anti-HA antibody and immunoblotted with anti-NPM (top) and anti-HA (middle) antibodies. Expression levels of NPM were assessed by immunoblotting of the total cell lysates with anti-NPM antibody (bottom). (C) Lysates of HeLa cells transfected with the mock or USP36 siRNA vectors were immunoblotted with anti-NPM, anti-USP36 and anti-{alpha}-tubulin antibodies.

 

Figure 5
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  		Fig. 5. USP36 deubiquitylates and stabilizes fibrillarin. (A) COS-7 cells transfected with Flag-fibrillarin with HA-tagged wild-type USP36 or USP36C131A for 2 days, and treated with MG132 for 8 hours. Their lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with FK2 (top) and anti-Flag (middle) antibodies. Closed arrowhead, open arrowhead and asterisk indicate non-, mono- and poly-ubiquitylated fibrillarin, respectively. Expression levels of the HA-USP36 constructs were assessed by immunoblotting of the total cell lysates with anti-HA antibody (bottom). (B-B'') HeLa cells transfected with Flag-fibrillarin and double-stained with anti-Flag (B) and anti-USP36 (B', red) antibodies. B'' is the merged image. Arrowheads indicate typical nucleoli. Asterisks indicate untransfected cells. Scale bar: 10 µm. (C) COS-7 cells transfected with Flag-fibrillarin with or without HA-USP36 for 2 days, and further cultured in the presence of CHX for 24 or 36 hours. Lysates of the cells were immunoblotted with anti-Flag, anti-HA and anti-{alpha}-tubulin antibodies.

 

Figure 6
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  		Fig. 6. USP36 is required for normal cell proliferation. (A) An equal number of HeLa cells were transfected with the mock or USP36 siRNA vectors for 6 days, trypsinized and the cell number counted. The relative number of siRNA-transfected cells compared with that of mock-transfected cells is shown as a percentage. Mean ± s.d. of a typical triplicate experiment is shown. (B) HeLa cells were treated with or without TNF-{alpha} and CHX (T + C) or transfected with USP36 siRNAs. Their lysates were immunoblotted with anti-PARP1, anti-USP36 and anti-{alpha}-tubulin antibodies. Closed and open arrowheads indicate uncleaved and cleaved PARP1, respectively. The reason for the reduced USP36 expression after treatment with TNF{alpha} and CHX is unclear.

 

Figure 7
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  		Fig. 7. USP36 is required for a normal nucleolar structure. (A-D'') HeLa cells transfected with the USP36 siRNA-1 (A-A'', B-B'', and D-D'') or mock (C-C'') vector and stained with anti-USP36 antibody (A-D) with anti-nucleolus (A',B') or anti-NPM (C',D') antibody. A''-D'' are merged images. Asterisks in A-A'' and B-B'' indicate USP36-depleted cells. Scale bars: 10 µm. (E,F) EM of HeLa cells transfected with mock vector (E) or USP36 siRNA-1 vector (F). N and arrowheads indicate the nucleoplasm and nucleoli, respectively. Insets show high-magnification images of the nucleoli. In nucleoli, the granular components (asterisks), and the dense fibrillar components (arrows) surrounding the fibrillar center, are indicated. Scale bars: 5 µm.

 

Figure 8
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  		Fig. 8. USP36 is required for biogenesis of rRNAs and ribosomes. (A) HeLa cells transfected with the mock or USP36 siRNA vectors incubated in the presence of [32P]orthophosphate for 45 minutes, and chased in the absence of [32P]orthophosphate for 30 or 90 minutes. Total RNA was isolated from an equal number of cells, separated by electrophoresis, and transferred to a nylon membrane. The membrane was exposed to X-ray film (top) or stained with methylene blue (bottom). The levels of 32P-labeled rRNAs increased during the chasing period, probably because intracellularly incorporated [32P]orthophosphate cannot be removed by rinsing with PBS. (B) Quantification of radioactivity of rRNAs in mock- and USP36 siRNA-1-transfected cells in A. The ratio of each processed rRNA to 47S pre-RNA is shown as a percentage. Closed circles, mock; open circles, USP36 siRNA-1. (C) HeLa cells were transfected with the mock or USP36 siRNA vectors, and cytoplasmic fractions were prepared from an equal number of cells. The fractions were subjected to 10-50% sucrose density gradient centrifugation and fractionated, and RNA was detected by measuring absorbance at 254 nm (A254). Positions of the 80S ribosome, 60S (closed arrowhead) and 40S (open arrowhead) ribosome subunits, and polysomes (P) are indicated.

 

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