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USP15 Regulates Neuromuscular Functionsthrough the Control of RNA Splicing
著者 KIM Jaehyun内容記述 この博士論文は内容の要約のみの公開(または一部
非公開)になっていますyear 2018その他のタイトル USP15によるRNAスプライシングを介在した神経筋の
機能制御メカニズムの解析学位授与大学 筑波大学 (University of Tsukuba)学位授与年度 2017報告番号 12102甲第8557号URL http://hdl.handle.net/2241/00152444
USP15 Regulates Neuromuscular Functions
through the Control of RNA Splicing
A Dissertation Submitted to
the Graduate School of Life and Environmental Sciences,
the University of Tsukuba
in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy in Science
(Doctoral Program in Biological Sciences)
Jeahyun KIM
i
Table of Contents
Abstract 1
Abbreviations 3
Introduction 5
Materials and Methods 7
Results
USP15 deficient mice exhibit ataxia-like symptoms 13
USP15 interacts with TUT1 and promotes its deubiquitination 14
USP15 deubiquitinates K63-linked polyubiquitin chins on TUT1 15
SART3 enhances interaction between USP15 and TUT1 16
USP15 promotes TUT1 subnuclear translocation 18
Loss of USP15 reduces U6-snRNA level in cerebellum 19
Loss of USP15 results in altered RNA splicing 19
USP4 regulates deubiquitination and translocations of TUT1 20
KLHL7 leads to relocation of TUT1 in nucleus 22
Discussion 24
Acknowledgements 26
References 27
Tables 33
Figures 39
1
Abstract
Ubiquitin system controls various physiological functions in cells by modulating
cellular processes such as protein degradation and signaling pathway. Recent studies
have implied that deubiquitinating enzyme (DUB) responsible for the removal of
ubiquitin from substrate is important as a regulator of neurological function. For
example, dysfunction or deficiency of certain DUBs results in neurodegenerative and
psychiatric disorders. Furthermore, it is also revealed that ubiquitin specific protease
15 (USP15), a member of large family of DUBs, is involved in several neurological
disorders including autism, ataxia, Parkinson's disease and glioblastoma, providing a
clue about the close relationship between USP15 and nervous system. However, the
detailed molecular mechanism of how USP15 works on nervous system is yet to be
elucidated.
In this study, I found that USP15 deficient mice show ataxia-like behavior
resulting from the functional defects of cerebellum and skeletal muscle. In
biochemical analysis, I revealed that USP15 deubiquitinates terminal uridylyl
transferase 1 (TUT1), which carries out polyuridylation on 3'-end of U6 snRNA, and
regulates TUT1 subnuclear localizations. SART3, which is a recycling factor of
U4/U6 di-snRNP, facilitates TUT1 deubiquitination as a substrate targeting factor of
USP15. Furthermore, USP4, highly homologous with USP15, also promotes
deubiquitination and translocation of TUT1 indicating that USP4 may play a same
role with USP15 in regulating TUT1. As to regulation of subnuclear translocation of
2
TUT1, KLHL7, an ubiquitin ligase, which interacts with USP15, also regulates
TUT1 translocation implying that KLHL7 is involved in USP15-TUT1 pathway.
In gene expression analysis, loss of USP15 reduces U6-snRNA level and
widely affects splicing pattern of multiple genes potentially related to neuromuscular
disorders, suggesting that USP15 is essential for proper neuromuscular functions
mediated by RNA splicing. Thus, this study may give a novel understanding between
RNA metabolism controlled by ubiquitin system and neuromuscular functions.
3
Abbreviations
ADP Adenosine diphosphate
ALS Amyotrophic lateral sclerosis
Cul Cullin
DUB Deubiquitinating enzyme
E1 Ubiquitin activating enzyme
E2 Ubiquitin conjugating enzyme
E3 Ubiquitin ligase
EDTA Ethylenediaminetetraacetic acid
FL Flag
IB Immunoblot
IP Immunoprecipitation
JAMMs JAB1/MPT/Mpv43 metalloenzymes
KLHL7 Kelch like protein 7
LSm Like-Sm
MEF Mouse embryonic fibroblast
NPM Nucleophosmin
OUT Ovarian tumor protease
PCR Polymerase chain reaction
PD Pull down
SART3 Squamous cell carcinoma antigen recognized by T cells 3
SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
4
SMA Spinal muscular atrophy
snRNA Small nuclear RNA
snRNP Small nuclear ribonucleic particles
TUT1 Terminal uridylyl transferase 1 (U6 snRNA-Specific)
Ub Ubiquitin
UCH Ubiquitin C-terminal hydrolases
USP Ubiquitin-specific proteases
WCL Whole cell lysates
WT Wild type
5
Introduction
Ubiquitin (Ub) system is responsible for regulating various cellular processes such as
protein degradation, DNA transcription, signal transduction and protein quality
control [1]. Ub is covalently attached to a target protein through a sequential action
of Ub activating enzyme (E1), Ub conjugating enzyme (E2), Ub ligase (E3), and is
removed from the target by deubiquitinating enzymes (DUBs). This reversible
reaction, which governs a balance of ubiquitination status of target proteins, is also
important for the control of nervous system functions including neurite growth [2],
synaptic transmission [3-5], receptor turnover [6, 7] and synaptic plasticity [8, 9] and
receive attention as a key regulator of nervous system functions.
DUBs have also attracted attention as therapeutic targets for
neurodegeneration. The importance of DUB function at nervous system was first
highlighted by specific mutation of DUB genes that link to several neurological
disorders [10, 11]. Furthermore, it has been established that dysfunction or
deficiency of DUBs results in disruption of synapse development and function,
neurodegenerative disorders and psychiatric disorders [12], indicating that DUBs
perform crucial functions in both the central and peripheral nervous system. DUBs
are composed of five distinct subfamilies: ubiquitin-specific proteases (USPs),
ubiquitin C-terminal hydrolases (UCHs), ovarian tumor protease (OTUs), Josephin
proteases, and JAB1/MPT/Mpv43 metalloenzymes (JAMMs) [12]. One of this
subfamily, USPs, which form a large family of deubiquitinating enzymes, is also
6
involved in several neurological disorders such as spinocerebellar ataxia type 1 [13],
Down’s syndrome [14, 15] and Parkinson’s disease [16, 17].
USP15 is a member of the USP family. Recent studies have revealed that
USP15 promotes oncogenesis in glioblastoma by activation of the TGF-β signaling
pathway [18] and the expression levels of both USP15 mRNA and protein decreases
in mouse models of spinocerebellar ataxia type 3 and Huntington’s disease [19].
These suggest that USP15 may play important roles in neuromuscular functions.
However, the detailed molecular mechanism of how USP15 functions in nervous and
muscle systems were yet to be elucidated.
In this study, I have focused on both physiological and molecular role of
USP15 in both neurons and muscle cell. Through the analyses of USP15 deficient
mice, I found that USP15 deficient mice exhibit motor dysfunctions, and lack of
USP15 induces global misregulation in RNA splicing. In molecular analyses, I
identified novel target of USP15. USP15 deubiquitinated TUT1, responsible for
polyuridylation of U6-snRNA, and regulated its subnuclear translocation. This
process was facilitated by SART3, substrate targeting factor of USP15. In these
analyses, I also found that USP4 and KLHL7 regulate TUT1 translocation implying
that these two proteins are involved in TUT1 regulation. These data, therefore,
suggest that USP15 is involved in the regulation of RNA splicing through the control
of TUT1, which is a key process that controls nervous and muscle system.
7
Materials and Methods
Rotarod performance test
2-month old wild-type and USP15 deficient mice were placed on rod. Rotation speed
was 5 r.p.m. at the beginning and then gradually accelerated to 40 r.p.m. The time to
fall from the rod was measured for 240 seconds.
Plasmids
LSms (1, 2, 4, 6 and 7), SART3 and TUT1 were amplified from HCT116 cells
cDNA library by using KOD plus DNA polymerase (TOYOBO) and inserted into
pcDNA3-Flag, pCS4-Myc, pCS4-HA, pCS4-EGFP vectors. SART3 deletion mutants
were amplified from SART3 wild-type plasmids and constructed likewise. The
pCAGEN-His-Ub (wild-type and mutants) was described previously [20]. pcDNA3-
Flag-USP15, pcDNA3-Myc-USP15, pcDNA3-Myc-USP15 D879A, pcDNA3-Flag-
USP4 were constructed previously in Tomoki Chiba laboratory (University of
Tsukuba). pcDNA-Myc-KLHL7 WT, pcDNA3-Myc-KLHL7 S150N, pcDNA3-
Myc-KLHL7 A153T and pcDNA3-Myc-KLHL7 A153V were describe previouly
[21]. The primers used in this study are described in Table 1.
Antibodies
For immunoblot analyses, anti-USP15 (Bethyl, A300-923A), anti-SART3
(Proteintech, 180251AP), anti-Tubulin (SIGMA, DM1A), anti-FLAG (SIGMA, M2),
anti-cMyc (Santa Cruz, 9E10), anti-GFP (MBL, 598) and anti-His (GE Helthcare,
8
27-4710-01) were used as primary antibodies. The peroxidase-conjugated anti-mouse
and anti-rabbit antibodies (SeraCare) were used as secondary antibodies.
Anti-Calbindin (SIGMA, CB-955) was used for immunohistochemistry as
primary antibodies. For immunofluorescence analyses, anti-Flag (SIGMA, M2), anti-
Flag (SIGMA, F7425), anti-HA (Roche, Roche) were used as primary antibodies.
Anti-NPM antibody was provided by Dr. Mitsuru Okuwaki [22]. Alexa Fluor 488
and 594 conjugated anti-rabbit, anti-rat and anti-mouse antibodies were used as
secondary antibodies.
Cell culture
Wild type and USP15 deficient MEFs, HEK293, HEK293T and HeLa cells were
cultured in Dulbecco's modified Eagle's medium (high glucose) (WAKO)
supplemented with 10% fetal bovine serum, 1% penicillin streptomycin (Gibco) in a
37°C incubator with 5% CO2.
Histology
Brains and skeletal muscles of 3-month-old and brains of 9-month-old mice were
perfused with 4% paraformaldehyde in phosphate buffered saline (PBS). Tissues
were fixed in the same fixative for 48 hours and then embedded in paraffin. Sections
were stained with Meyer's Hematoxylin and Eosin or subjected to
immunohistochemical analyses.
Immunohistochemistry
9
Deparaffinized tissue specimens were immersed in 0.01 M citrate buffer (10mM
Citric Acid, 0.05% Tween 20, pH 6.0) and boiled in microwave oven for 10 minutes.
After antigen retrieval, tissue sections were blocked for an hour in 3% bovine serum
albumin (BSA) in TBST (25 mM Tris-HCl (pH 7.5), 0.14 M NaCl, 0.1% TritonX-
100) and incubated with a primary antibody, mouse anti-Calbindin antibody diluted
1/300 in TBST for overnight at 4°C. Following wash with TBST, tissue sections
were incubated with a secondary antibody (Alexa Fluor 594 anti-mouse IgG) diluted
1/500 in TBST for an hour at room temperature. Tissue specimens were observed
using a fluorescence microscope (Keyence, BIOREVO BZ-9000).
Immunoprecipitation
Cells were lysed with ice-cold lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl,
1 mM EDTA, 0.5% NP-40, 1 mM DTT) and centrifuged at 14,000 r.p.m. for 10
minutes. The supernatant was subjected to immunoprecipitation with anti-USP15 or
anti-cMyc antibodies and protein G agarose beads (Thermo Scientific), or anti-Flag
M2-agarose beads (SIGMA). Samples were incubated at 4°C overnight. The beads
were then washed with lysis buffer. The protein samples were added to 4 X SDS
sample buffer (250 mM Tris-HCl (pH 6.8), 40% Glycerol, 10% SDS, 0.04%
bromophenol blue, 20% β-mercaptoethanol), boiled for 3 minutes and subjected to
immunoblot analysis.
His-Ub Pull down assay
10
Cells were washed with PBS and lysed in extraction buffer (6M guanidinium-HCl,
50 mM sodium phosphate buffer (pH 8.0), 300 mM NaCl and 5 mM imidazole). Cell
lysates were sonicated for 30 seconds on ice, centrifuged and then incubated with
Talon metal affinity resin (Clonetech) at 4°C overnight. The precipitants were
washed with buffer (50 mM sodium phosphate buffer (pH 8.0), 300 mM NaCl and 5
mM imidazole) and subjected to immunoblot analysis.
Immunoblot
The protein samples after immunoprecipitation and his-tagged pull down assay were
separated by SDS-PAGE, transferred to PVDF membranes (Millipore). Membranes
were incubated overnight at 4°C with primary antibodies. The proteins on membrane
were detected with HRP-conjugated secondary antibodies and chemiluminescence
reagent (Amersham ECL Prime Western Blotting Detection Reagents, GE
Healthcare).
Immunocytochemistry
HeLa cells were cultured on cover slips and after 18 hours, transfected with indicated
plasmids. The cells were fixed with 4% paraformaldehyde in PBS for 10 min at room
temperature. The cells on coverslips were blocked in 0.4% Triton X-100 in blocking
solution (3% BSA in PBS) for 30 min at room temperature, and then incubated with
primary antibodies diluted in blocking solution for an hour or overnight at 4 °C.
After washing with PBS, the cells were incubated with secondary antibodies diluted
in blocking solution for 30 minutes at room temperature. Nuclei were stained with
11
Hoechst 33342 (Life Technologies). The coverslips were then mounted onto slides
using the Fluoromount Plus mounting solution (Diagnostic BioSystems). Images
were obtained using a fluorescence microscope (Keyence model BZ-9000).
Quantitative real-time PCR (qPCR)
Total RNAs from wild-type and USP15 deficient cerebellum, cortex, skeletal muscle,
liver, spleen, kidney, heart and MEFs were prepared by ISOGEN II (NIPPON
GENE). The cDNA were synthesized by SuperScript III CellsDirect cDNA
Synthesis Kit (Life Technologies) using random hexamer primer. qPCR was
performed with THUNDERBIRD SYBR qPCR Mix (TOYOBO). The data were
analyzed using Thermal Cycler Dice Real Time System Software (TAKARA).
Following Primers were used.
5.8 S rRNA qPCR Forward, 5'-CGGCTCGTGCGTCGAT-3’;
5.8 S rRNA qPCR Reverse, 5'-CCGCAAGTGCGTTCGAA -3’;
U6-snRNA qPCR Forward, 5'-GCTTCGGCAGCACATATACTAAAAT-3';
U6-snRNA qPCR Reverse, 5'-ACGAATTTGCGTGTCATCCTT-3'.
Exon array
One-month-old mice of wild type and USP15 deficient mice (n=3) were euthanized
using carbon dioxide. Their cerebellum and skeletal muscles were then dissected and
snap frozen in liquid nitrogen. Total RNAs were extracted from each tissue using
ISOGEN II (NIPPON GENE) according to the manufacture’s instructions.
Fragmented and labelled total RNA of each sample were hybridized on Affymetrix
12
GeneChip mouse exon 1.0 ST arrays. After hybridization, each probe array was
washed and stained with Affymetrix GeneChip Fluidics Station 450 and scanned by
Affymetrix GeneChip Scanner 3000. Data were analyzed with GeneSpring 12.6
Software and filtered by more than Splicing Index 0.5 and with P-value<0.05.
Splicing Index was calculated with following calculation:
Splicing index = log2 (NIi1/NIi2)
NIij = Eij/Gj
NIij means normalized intensity (NI) for exon i in experiment j. Eij is the estimated
intensity level for exon i in experiment j. Gj is the estimated gene intensity.
13
Results
USP15 deficient mice exhibit ataxia-like symptoms.
Although USP15 appears to be related with neuromuscular disorders [19], little is
known about the detailed function of USP15 in the nervous systems. To understand
the physiological roles of USP15 in the neuromuscular system, I analyzed USP15
deficient mice. USP15 deficient mice were much smaller than wild-type mice in
body size (Figure 1). It also showed hind limb clasping reflexes in tail suspension
test, which is a typical phenotype observed in mice with ataxia (Figure 2). To further
investigate whether lack of USP15 impairs motor ability, rotarod performance test
was performed. USP15 deficient mice easily lost balances and tended to grip the rod
instead of walking on the rotating rod, and eventually fell to the ground earlier than
wild-type ones (Figure 3). These results indicate that lack of USP15 leads to motor
disorder.
It is widely known that dysfunctions of cerebellum or skeletal muscle may
cause ataxia-like symptoms. Therefore, I next examined the tissue specimens derived
from cerebellum and skeletal muscle of USP15 deficient mice. In USP15 deficient
cerebellum, abnormal branching and position of lobules were observed (Figure 4). In
addition, immunohistochemical analyses using Purkinje cell marker, anti-Calbindin
antibody, revealed that loss of Purkinje cells, which contribute to motor coordination,
was observed in USP15 deficient cerebellum (Figure 5). In skeletal muscle, 70%
reduction in muscle fiber thickness was observed in USP15 deficient mice (Figure 6).
14
Theses data shows that USP15 is important for the regulation of cerebellar and
muscular morphogenesis and lack of USP15 causes motor dysfunction.
USP15 interacts with TUT1 and promotes its deubiquitination.
In order to figure out the mechanism of how the loss of USP15 leads to motor
dysfunction, I explored deubiquitination substrates of USP15. Mass spectrometry
analysis of USP15 interacting proteins identified several proteins related to U6-
snRNP, which is a core component of spliceosome and carries out mRNA splicing
(Table 2) implying that USP15 may be involved in spliceosomal functions. LSm
proteins assists the association of U4/U6 di-snRNP by assembly of LSm ring [23].
SART3 is known as a recycling factor of U4/U6-snRNP. It facilitates disassembly of
U4/U6 di-snRNP by recruiting USP4 to its deubiquitination substrates Prp3, a
component of U4-snRNP [24-26]. TUT1 is responsible for polyuridylation on 3'- end
of U6-snRNA [27]. To further investigate the interaction between USP15 and these
U6-snRNP related proteins, co-IPs in HEK293 cells were performed. I first
examined whether LSm proteins bind to USP15. Although several LSm proteins
were identified as the binding targets by mass spectrometry, co-IP with USP15 could
not be seen (Figure 7). I next tested whether SART3 bind to USP15. Protein lysates
from wild-type and USP15 deficient MEFs as a negative control, were
immunoprecipitated with anti-USP15 antibodies and immunoblotted with both anti-
USP15 and anti-SART3 antibodies. Endogenous SART3 was co-immunoprecipitated
with endogenous USP15, indicating that USP15 binds to SART3 (Figure 8). Lastly, I
investigated if TUT bind to USP15 by expressing Myc-USP15 and GFP-TUT1 in
15
HEK293 cells. Myc-USP15 was immunoprecipitated and immunoblotted with anti-
Myc and anti-GFP antibodies. GFP-TUT1 was co-immunoprecipitated with Myc-
USP15, indicating that USP15 binds to TUT1 (Figure 9).
Since USP15 binds to TUT1, I then investigated whether TUT1 is a substrate
of USP15 by His-Ub pull down assay. HEK293 cells were transfected with Flag-
TUT1, His-Ub and either Myc-USP15 or mock. Ubiquitinated proteins were pulled-
down with Talon metal affinity resin and immunoblotted with anti-Flag, anti-Myc
and anti-His antibodies. Ubiquitinated TUT1 was detected in the absence of USP15
overexpression. On the other hand, overexpression of Myc-USP15 decreased the
amount of ubiquitinated TUT1, while that of Myc-USP15 D879A mutant that lacks
its enzyme activity did not, suggesting that USP15 deubiquitinates TUT1. Together,
theses data suggest that TUT1 is the target substrate of USP15 (Figure 10).
USP15 deubiquitinates K63-linked polyubiquitin chains on TUT1.
To figure out which polyUb chain on TUT1 is targeted by USP15, I used his- wild-
type and mutant (K48R, K63R, 48K and 63K) Ub expression plasmids for his-tagged
pull down assay. As polyUb chains could be formed via lysine residues, mutant Ub
of lysine 48 to arginine (K48R) or lysine 63 to arginine (K63R) are unable to form
polyUb chains via lysine 48 or 63 linkages respectively. Ub of every lysine mutated
to arginine except lysine 48 (K48) or lysine 63 (K63) could form only polyUb chains
via lysine 48 or 63 linkages. HEK293 cells expressing Flag-TUT1, Myc-USP15 and
either WT or mutant His-Ub were pulled-down with Talon metal affinity resin and
immunoblotted with anti-Flag, anti-Myc and anti-His antibodies. TUT1
16
deubiquitination by USP15 was observed in overexpression of WT, K48R and 63K
Ub, but not that of K63R and 48K (Figure 11). These results indicate that USP15
deubiquitinates K63-linked polyUb chains on TUT1. Unlike K48-linked polyUb that
is related to degradation by proteasome, K63-linked polyUb is involved in signaling
pathway such as inflammation and translocation. Thus, these data suggest that
USP15 controls the function of TUT1 through its deubiquitination.
SART3 enhances interaction between USP15 and TUT1.
Previous studies revealed that SART3 binds to USP4 and guide it to Prp3, and
thereby promotes deubiquitination of Prp3 as a substrate targeting factor of USP4
[24]. I hypothesized that SART3 may play an equal role in regulating USP15. To test
this, I first investigate whether SART3 binds to TUT1 by expressing Flag-TUT1 and
Myc-SART3 in HEK293 cells. Myc-SART3 was immunoprecipitated and
immunoblotted with anti-Myc and anti-GFP antibodies. As expected, TUT1 was co-
immunoprecipitated with SART3 indicating that SART3 also binds to TUT1 (Figure
12). Next, I examined interaction between USP15 and TUT1 with or without
overexpression of SART3. HEK293T cells expressing Flag-TUT1, GFP-SART3 and
Myc-USP15 were immunoprecipitated with anti-Myc antibody and immunoblotted
with anti-Flag, anti-GFP and anti-Myc antibodies. Though Co-IP of Flag-TUT1 with
Myc-USP15 could be seen without GFP-SART3 overexpression (Figure 9 and
Figure 13, lane 6), it increased in GFP-SART3 overexpression (Figure 13, lanes 7
and 8). This suggests that SART3 is a substrate targeting factor of USP15
17
To characterize the region of SART3 where TUT1 binds, SART3 deletion
mutants (SART31~649 and SART3660~963) were used for co-IP experiment. Myc-
SART3 WT or deletion mutants were co-expressed with Flag-TUT1 in HEK293T
cells. Cell lysates were immunoprecipitated with anti-Flag antibody and
immunoblotted with anti-Myc and anti-Flag antibodies. Flag-TUT1 co-
immunoprecipitated Myc-SART3 and Myc-SART3660~963, but did not Myc-
SART1~649 (Figure 14). It indicates that TUT1 binds to SART3 C-terminal region
(660~963 amino acids region). Previous study reported that USP15 binds to SART3
278~649 amino acids region [28]. Therefore, USP15 and TUT1 bind to different
regions of SART3 and I thought SART3278~649 and SART3660~963 deletion mutants
could be used as a dominant negative mutant.
Next, I investigated the interaction between TUT1 and USP15 with or
without SART3 WT and deletion mutants by overexpressing GFP-TUT1, Flag-
USP15 and either Myc-SART3 or deletion mutants in HEK293T cells. Flag-USP15
was immunoprecipitated and immunoblotted with anti-GFP, anti-Myc and anti-Flag
antibodies. Co-IP of GFP-TUT1 with Flag-USP15 did not increase in Myc-SART3
deletion mutants (either SART3 227~649 or SART3 660~963) overexpression, like in
Myc-SART3 WT overexpression (Figure 15). This indicates that SART3 deletion
mutants could not increase the interaction between USP15 and TUT1.
To further investigate the regulation of USP15 by SART3, his-Ub pull down
assay was performed by expressing Flag-TUT1, Myc-USP15, His-Ub and either
Myc-SART3 WT or Myc-SART3660~963 in HEK293 cells. Ubiquitinated proteins
were pulled-down with Talon metal affinity resin and immunoblotted with anti-Flag,
18
anti-Myc and anti-His antibodies. Myc-SART3 WT overexpression increased TUT1
deubiquitination by USP15, but Myc-SART3660~936 overexpression did not (Figure
16). These results show that SART3 enhances interaction between USP15 and TUT1
by binding to both proteins, and lead to TUT1 deubiquitination as a substrate
targeting factor of USP15.
USP15 promotes TUT1 subnuclear translocation.
Next, to figure out the physiological role of deubiquitination of TUT1, I investigated
subcellular localization of TUT1. HeLa cells were transfected with Flag-TUT1 and
co-stained with anti-NPM and anti-Flag antibodies. Flag-TUT1 was co-localized
with NPM, showing that TUT1 is mainly localized in nucleolus (Figure 17). As
USP15 deubiquitinates TUT1 and SART3 promotes this process, I then examined if
overexpression of WT or mutants USP15 and SART3 affects TUT1 localization.
Flag-TUT1 was co-expressed with Myc-USP15 or Myc-USP15 D879A or both Myc-
USP15 and HA-SART3 or Myc-SART3660~963 in HeLa cells, and stained with anti-
Flag and anti-Myc antibodies. Flag-TUT1 was relocated to nucleoplasm in Myc-
USP15 only or both Myc-USP15 and HA-SART3 overexpression, but was still
localized within the nucleolus in Myc-USP15 D879A and Myc-SART3660~963
overexpression (Figure 18A). Furthermore, Myc-USP15 was localized in nucleus in
HA-SART3 overexpression indicating that SART3 recruits USP15 to nucleus where
TUT1 is localized. The ratio of TUT1 localized in nucleolus (Type1) increased in
both Myc-USP15 and HA-SART3 overexpression more than in Myc-USP15
overexpression indicating that SART3 regulates TUT1 translocation (Figure 18B). It
19
is interesting that TUT1 localized in nucleolus ratio (Type1) pattern shown in
quantification data is similar to the ubiquitinated TUT1 ratio pattern seen in the his-
tagged pull down assay (Figure 16 and 18B). Together, these results indicate that
USP15 and SART3 promote TUT1 translocation from nucleolus to nucleoplasm
through its deubiquitination.
Loss of USP15 reduces U6-snRNA level in cerebellum.
As TUT1 polyuridylates 3'- end of U6-snRNA, I hypothesized that lack of USP15
affects U6-snRNAs stability. Therefore, I analyzed the U6-snRNA level, a core
spliceosomal component, via quantitative polymerase chain reaction (qPCR). Total
RNAs were purified from WT and USP15 deficient cerebellum, cortex, skeletal
muscle, liver, spleen, kidney, heart and MEFs. The RNAs were reverse transcribed,
and the U6-snRNA levels were measured by qPCR. The U6-snRNA level decreased
only in USP15 deficient cerebellum. It was 0.8 fold lower compared to WT (Figure
19A). On the contrary, The U6-snRNA levels in other tissues did not decrease
(Figure 19 B, C, D, E, F G and H). Therefore, it is likely that the mechanisms that
underlie the regulation of spliceosomal components mediated by USP15 may be
tissue-specific.
Loss of USP15 results in altered RNA splicing.
Since the loss of USP15 induces lower U6-snRNA level in cerebellum, USP15 could
be involved in regulation of RNA splicing machinery. It has been known that
misregulation of RNA splicing causes neuromuscular disease such as amyotrophic
20
lateral sclerosis and spinal muscular atrophy (Gubitz et al., 2004; Kwiatkowski et al.,
2009; Vance et al., 2009). Therefore, ataxia-like symptoms observed in USP15
deficient mice may be caused by abnormal RNA splicing.
To determine whether the loss of USP15 affects RNA splicing, I performed
exon array analysis, which can measure the relative levels of individual exons. Total
RNAs extracted from the cerebellums and skeletal muscles of three pairs of one-
month-old female littermates were used for exon arrays. From the datasets, I
identified a large number of genes with exon transcripts that were altered
significantly in USP15 deficient mice compared to wild-type mice. This analysis
identified 246 genes in cerebellum and 206 genes in skeletal muscle (Table 3, 4, 5
and 6: data only shows the 10 genes with the highest splicing index score). These
results demonstrated that RNA splicing in USP15 deficient cerebellum and skeletal
muscle was widely changed in multiple genes which may include the candidate
genes related to neuromuscular diseases. Thus, loss of USP15 appears to trigger a
huge change in mRNA splicing critical for nervous and muscle systems and may
result in failure of neuromuscular functions. Interestingly, genes altered in RNA
splicing due to the lack of USP15 are quite different between cerebellum and skeletal
muscle. These data imply that RNA splicing modulated by USP15 may be tissue-
specific, and this specificity enables to maintain proper neuromuscular functions via
regulation of RNA splicing.
USP4 regulates deubiquitination and translocations of TUT1.
21
Given that USP4 is highly homologous with USP15 and recruited to nucleoplasm by
SART3 like USP15 [24, 28], it may be possible that USP4 also targets TUT1. To test
this assumption, I investigated interaction between USP4 and TUT1 by expressing
GFP-TUT1, Flag-USP4 and either Myc-SART3 WT, Myc-SART3278~649 or Myc-
SART3660~963 in HEK293T cells. Flag-USP4 was immunoprecipitated with anti-Flag
and immunoblotted with anti-GFP, anti-Myc and anti-Flag antibodies. Co-IP of GFP-
TUT1 with Flag-USP4 could be seen indicating that TUT1 binds to USP4 (Figure 20,
lane 9). Interestingly, Myc-SART3 overexpression decreased interaction of GFP-
TUT1 with Flag-USP4. However, either Myc-SART3278~649 or Myc-SART3660~963
overexpression did not change it (Figure lanes 20, 10, 11 and 12) suggesting that
SART3 prefers to recruit USP15 than USP4 in regulating TUT1.
I then examined if TUT1 is a deubiquitination target of USP4 by His-Ub pull
down assay. HEK293 cells were transfected with Myc-TUT1, Flag-USP4, His-Ub
and either GFP-SART3 WT or GFP-SART3660-963. Ubiquitinated proteins were
pulled-down with Talon metal affinity resin and immunoblotted with anti-Flag, anti-
Myc, anti-GFP and anti-His antibodies. TUT1 was deubiquitinated in Flag-USP4
overexpression, which was stronger with GFP-SART3 overexpression. On the other
hands, GFP-SART3660~963 overexpression did not lead to TUT1 deubiquitination
(Figure 21). Taken together, these data show that USP4 is also responsible for TUT1
deubiquitination and SART3 functions as a substrate targeting factor of USP4 for
TUT1.
Since both USP15 and USP4 are in charge of TUT1 deubiquitination, I
assumed that both USP15 and USP4 overexpression could increase TUT1
22
deubiquitination much more. HEK294 cells were transfected Myc-TUT1, Flag-
USP15, Flag-USP4 and His-Ub. Ubiquitinated proteins were pulled-down with
Talon metal affinity resin and immunoblotted with anti-Flag, anti-Myc and anti-His
antibodies. TUT1 deubiquitination increased slightly in both Flag-USP15 and Flag-
USP4 overexpression, compared to each Flag-USP15 and Flag-USP4 overexpression
individually (Figure 22). It indicates that both USP15 and USP4 can deubiquitinate
TUT1. I also investigated the subnuclear localization of TUT1 with USP4. HA-
TUT1 was co-expressed with Flag-USP4 in the presence or absence of Myc-SART3
or Myc-USP15 in HeLa cells, and stained with anti-HA and anti-Flag antibodies.
Flag-USP4 expression translocated HA-TUT1 to nucleoplasm like USP15 (Figure
23A). Co-overexpression with Myc-SART3 or Myc-USP15 also induced TUT1
translocation (Figure 23B). Thus, these results suggest that USP15 and USP4 carry
out the same role for the regulation of TUT1 at least in cells.
KLHL7 leads to relocation of TUT1 in nucleus.
In our preliminary study, KLHL7 is shown to interact with USP15 and several
proteins involved in mRNA splicing machinery. KLHL7 is a component of Cul3-
based ubiquitin ligase [21] and its mutations cause retinitis pigmentosa [29, 30]. As
KLHL7 interacts with USP15 and seems to be related to RNA splicing machinery, I
assumed that KLHL7 might be involved in USP15-TUT1 regulatory pathway. To
test this assumption, I analyzed TUT1 subnuclear localization. Flag-TUT1 was co-
expressed with Myc-KLHL7 WT or disease-causative mutants (A153T, A153V and
S150N) in HeLa cells, and stained with anti-Myc and anti-Flag antibodies. TUT1
23
was localized in nucleoplasm with expression of KLHL7 WT and S150N mutant. On
the contrary, TUT1 still stayed in nucleolus with expression of KLHL7 A153T and
A153V mutants (Figure 24). Given that KLHL7 A153 mutations inhibit Cul3
interaction but A150N does not [21], these data indicate that KLHL7 activity, as an
ubiquitin ligase seems to be important for TUT1 translocation to nucleoplasm.
24
Discussion
Alternative mRNA splicing plays an important role in generating enormous
proteomic diversity. It allows eukaryotic cell to produce a huge number of proteins
from the limited number of genes (20,000~25,000 in the human genome) through
selective elimination of introns and exon joining and may thereby contribute to
tissue-specific functions. In brain, alternative splicing is involved in neuronal
functions through the regulation of gene expression, which acts in the synapse such
as neurotransmitter receptors, cation channels, adhesion and scaffold proteins [31].
Given the importance of alternative mRNA splicing in regulating neuronal functions,
it is not surprising that disruption of RNA splicing leads to neuronal dysfunction, and
thereby neurodegenerative disorder. Indeed, recent studies have indicated that
disruption and misregulation of RNA splicing results in neuromuscular disorders
such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA) [32-
34]. However, the biological mechanisms of how the failure in RNA splicing leads to
neuromuscular diseases remains unclear.
In this study, I report that TUT1 is the target substrate of USP15. USP15
deubiquitinated K63-linked polyUb chains on TUT1. Given that K63-linked polyUb
is mainly involved in signaling pathway, not degradation by proteasome, USP15
could regulate TUT1 functions through its deubiquitination. Indeed, USP15
promotes TUT1 subnuclear translocation, suggesting that deubiquitination of K63-
linked polyUb chains. TUT1 is important for translocation to nucleoplasm from
nucleolus. Since TUT1 is responsible for uridylation of 3'-region of U6-snRNA, it is
25
possible that USP15 may be associated with spliceosomal functions through
regulation of TUT1 and, furthermore, affect on RNA splicing. Indeed, lack of USP15
caused reduction in U6-snRNA level in cerebellum and a global alteration in RNA
splicng of various genes indicating that lack of USP15 could lead to misregulation on
RNA splicing machinery. Since dysfunction of alternative mRNA splicing results in
neuromuscular disorder, it is likely that disruption of USP15-TUT1 pathway, which
arouses the misregulation on RNA splicing machinery, induces accumulation of mis-
spliced forms of various genes that could affect neuromuscular functions and
therefore, eventually results in motor disorder shown in USP15 deficient mice.
It is interesting that RNA splicing enacts on every cells but disruption of
RNA splicing mainly results in neuromuscular disorder. In USP15 deficient mice,
USP15 is defective on every tissue but the symptom is quite tissue-specific. In this
study, I found that not only USP15 but also USP4 targeted TUT1 implying that
USP15 and USP4 may assist each other's deubiquitination functions. Furthermore,
SART3 and KLHL7 also regulate TUT1. It is possible that activity and function of
USP15, USP4, SART4 and KLHL7 may differ in each tissue, and thereby TUT1
could be regulated in a tissue-dependent manner. This notion may provide a clue
about tissue-specificity of neuromuscular disorder resulted from misregulation of
RNA splicing.
26
Acknowledgements
I express my deep thanks to Prof. Tomoki Chiba and Assistant Prof. Fuminori
Tsuruta for their persistent mentoring and guidance throughout this research. I also
thank Dr. Tohru Natsume (AIST) for mass spectrometry data and all the members of
Chiba laboratory for helpful discussions and technical supports.
27
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34
Table 2. USP15 interacting proteins identified by mass spectrometry.
* Proteins involved in RNA spliceosomal functions
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
Figure 17. TUT1 is localized at nucleolus.
HeLa cells transfected with Flag-TUT1 were stained with anti-NPM (nucleophosmin)
and anti-Flag antibodies and Hoechest 33342. Scale bar, 20 µm.
56
57
58
59
60
61
62
A B
Figure 24. Overexpression of Wild-type KLHL7 and KLHL7 SN mutant
induces relocation of TUT1 from nucleolus to nucleoplasm.
A, Fluorescent images of HeLa cells expressing Myc-TUT1 together with each
KLHL7 wild-type and dieses causative mutant plasmids. Scale bar: 20 µm. B,
Quantification of the data in (A). Subnuclear localization of Myc-TUT1 was
examined. Cells that express Flag-TUT1 in nucleolus (Type 1) and in diffusely
distribution (Type 2) were counted. n=30 ~ 34.
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