1. Das_Figure S1 USP15 USP4 USP11 49% 60% 39 % 71 % 52 % 54 %
963
952
seq. identity
49%
60%
39 %
71 %
52 %
54 %
DUSP
UBL
UCH
Supplementary Figure S1. USP15 shares high sequence homology with USP4
Schematic diagram of the domain organization of USP4, USP15 and USP11. They share the same domain organization and sequence similarity.

2. Das_Figure S2
siControl
siUSP15-3
siUSP15-2
siUSP15-1
Supplementary Figure S2. USP15 knockdown causes mitotic error
HeLa cells were transfected with control siRNA or three different siRNAs targeting different regions of the USP15 gene. The number of mitotic cells with errors in chromosome segregation or spindle formation was counted at 40X magnification with a fluorescence microscope. The data are shown as the mean ± S.D. from three independent experiments (n > 300 cells per experiment).

3. Das_Figure S3 A B SART3 actin
Cells with mitotic error (%)
siControl
siSART3-1
siSART3-2 B
actin
siControl
siSART3 -1
siSART3-2
SART3
Supplementary Figure S3. Depletion of SART3 induces mitotic defects
HeLa cells were transfected with control siRNA or SART3 siRNA. (A) The number of mitotic cells with errors in chromosome segregation or spindle formation was counted at 40X magnification with a fluorescence microscope. The data are shown as the mean ± S.D. from three independent experiments (n > 300 cells per experiment). (B) For confirmation of the depletion, cell lysates were immunoblotted with anti-SART3 and anti-b-actin.

4. Das_Figure S4 A B C D E GST pulldown GST GST-USP41-230 GST-USP151-226
SART31-691 72 95 55 43 34 26
Input
5% Input
IP : HA
Myc-SART3
HA-USP15
Myc HA
1-226
5% Input
IP : HA
Myc-SART3
HA-USP15
Myc HA
130
110
130 95 72 55 43 34 28
130
110 D 8
119
131
221
257
441
529
901
952 94
272
310
600
874
963
649
705
USP15
SART3
DUSP
UBL
UCH
HAT-N
NLS
RRM
LSM
HAT-C E
IP : HA
5% Input
Control
1-963
1-705
Control
1-963
1-705
HA-SART3
110
USP15
130 95 72 55 43 34 28 HA
Actin

5. Supplementary Figure S4. USP15 interacts with SART3
(A) SART3 binds to USP15 in cells. HeLa cells were transfected with Myc-SART3 and HA-USP15. HA- USP15 was purified on HA-agarose, and coprecipitating Myc-SART3 was detected by anti-Myc antibodies.
(B) N-terminus of USP15 is required for interaction with SART3. GST, GST-USP or GST-USP was immobilized on glutathione beads and incubated with purified SART Bound proteins were separated in SDS-PAGE and detected by Coomassie staining.
(C) HeLa cells were transfected with Myc-SART3 and HA truncations of USP15. HA tagged truncations of USP15 were purified on HA-agarose, and coprecipitated Myc-SART3 was detected by anti-Myc antibodies.
(D) Schematic overview of USP15 and its interaction with SART3. USP15 consists of the N-terminal DUSP- UBL domain and C-terminal UCH domain with inserted UBL domain. On the other hand SART3 consists of HAT-N, HAT-C of 12 HAT repeats, one NLS, two RNA recognition motifs (RRM), and one LSM domain as described in previous report (22). The DUSP-UBL domain of USP15 interacts with the HAT-C domain of SART3. The interaction regions are marked by arrow.
(E) SART3 interacts with USP15 through the HAT-C domain. HeLa cells were transfected with HA-tagged truncations of SART3. HA-tagged truncations of SART3 were purified on HA-agarose, and coprecipitated endogenous USP15 was detected by anti-USP15 antibodies.

6. Das_Figure S5 A B
Myc-PRP4B kinase
His-Ubi
HA-USP15
HA-USP15C269A
HA-PRP19
PRP4B kinase
Myc-USP39
His-Ubi
HA-USP15
HA-USP15C269A
HA-PRP19
USP39 C D
Myc-PRP8 C-term
His-Ubi
HA-USP15
HA-USP15C269A
HA-PRP19
denat. NiNTA-PD
PRP8 C-termUBI
His-Ubi
Myc-PRP4
HA-PRP19
HA-USP15
denat. NiNTA-PD
PRP4UBI
Supplementary Figure S5. Identification of USP15 substrate
Substrate candidates were overexpressed with His-ubiquitin, PRP19 and USP15. (A, B) Twenty-four hours after transfection, cell lysates were separated in SDS-PAGE and immunoblotted with anti-Myc antibodies. (C, D) Covalently modified proteins were purified on NiNTA-agarose under denaturing conditions. Ubiquitinated proteins were detected by anti-Myc antibodies.

7. Das_Figure S6 A B PRP31 SART3
SART PRP
SART PRP
n = 1.07
KD = 5.16 mM B
NOP
Colied coil 85
120
181
215
333
499
PRP31 94
272
310
600
874
963
649
705
SART3
HAT-N
NLS
RRM
LSM
HAT-C
Supplementary Figure S6. SART3 interacts with PRP31 directly
(A) ITC analysis of the interaction between SART and PRP or PRP There is no binding data for SART and PRP (left), while the binding between SART and PRP (right) is endothermic, and the analysis of the data shows a KD of 5.16 μM. (B) Schematic overview of PRP31 and its interaction with SART3. PRP31 consists of NOP and a coiled coil domain. On the other hand SART3 consists of HAT-N, HAT-C, NLS, two RNA recognition motifs (RRM), and one LSM domain. C-terminus of PRP31 interacts with the N-terminus of SART3. The interaction regions are marked by an arrow.

8. Das_Figure S7 A
MYC
DAPI
Merge
Myc-SART3
HA-USP15
+ HA-USP15 HA B
MYC
DAPI
Merge HA
Myc-PRP31
+ HA-SART3
Supplementary Figure S7. SART3 causes nuclear translocation of USP15
(A) SART3 causes nuclear translocation of USP15. HeLa cells were transfected with Myc-SART3 alone, HA-USP15 alone, or coexpression of Myc-SART3 and HA-USP15. After 48 h, cells were fixed with 4% formaldehyde, and incubated with anti-HA and anti-Myc antibodies, followed by secondary goat anti-rabbit antibodies coupled to Alexa488 and goat anti-mouse antibodies coupled to Alexa546. The nucleus was counterstained with DAPI. The intracellular localization of Myc-SART3 (green) or HA-USP15 (red) was analyzed with a confocal microscope. (B) PRP31 colocalizes with SART3. HeLa cells were transfected with Myc-PRP31, in the presence or absence of HA-SART3. The cells were immunostained with anti-HA and anti-Myc antibodies followed by Alexa488-conjugated anti-rabbit antibodies and Alexa546-conjugated anti-mouse antibodies. The nucleus was stained with DAPI. The intracellular localization of Myc-PRP31 (green) or HA-SART3 (red) was analyzed by confocal microscopy.

9. Das_Figure S8 A B USP4 USP15 L1 L2
963
952
223
255
441
758
227
297
483
777
DUSP
UBL
UCH
Linker 1
Linker 2 B L1 L2
Supplementary Figure S8. Alignment of the amino acid sequence of linker 1 and linker 2 in USP4 and USP15
(A) Schematic diagrams of USP4 and USP15. Linker 1(L1) and linker 2 (L2) of each protein are shown in black boxes. (B) Sequence alignment of linker 1(L1) and linker 2 (L2) of USP4 and USP15. The figure was generated with the ClustalX and Genedoc programs.

10. Das_Figure S9 A B
USP USP41-230 72
130 55 43 34 26
GST-USP
GST
coomassie
western
Input
Myc-USP4
Supplementary Figure S9. Interaction between USP15 and USP4 is not direct.
(A) ITC analysis of the interaction between USP and USP There is no binding data for USP and USP41-230(left) (B) Control GST or GST-USP was immobilized on glutathione beads and incubated with in vitro transcribed and translated Myc-USP4. Bound proteins were separated in SDS-PAGE and detected by western blotting and Coomassie staining.