From the Howard Hughes Medical Institute and Department of Biochemistry and Biophysics, University of Pennsylvania, School of Medicine, Philadelphia, Pennsylvania 19104
Received for publication, October 6, 2000, and in revised form, November 22, 2000
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ABSTRACT |
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The motor neuron degenerative disease spinal
muscular atrophy is caused by reduced expression of the survival
motor neuron (SMN) protein. Here we report a genetic system developed
in the chicken pre-B cell line DT40, in which the endogenous
SMN gene is disrupted by homologous recombination, and SMN
protein is expressed from a chicken SMN cDNA under control of a
tetracycline (tet)-repressible promoter. Addition of tet results in
depletion of SMN protein and consequent cell death, which directly
demonstrates that SMN is required for cell viability. The tet-induced
lethality can be rescued by expression of human SMN, indicating that
the function of SMN is highly conserved between the two species. Cells
expressing low levels of SMN display slow growth proportional to the
amount of SMN they contain. Interestingly, the level of the
SMN-interacting protein Gemin2 decreases significantly following
depletion of SMN, supporting the conclusion that SMN and Gemin2 form a
stable complex in vivo. This system provides a powerful
setting for studying the function of SMN in vivo and for
screening for potential therapeutics for spinal muscular atrophy.
Spinal muscular atrophy
(SMA),1 an autosomal
recessive disease with characteristics of motor neuron degeneration and
muscle atrophy, is a common childhood genetic disorder and the most
frequent genetic cause of infant mortality (1-3). Based on the age of onset and the severity of the disease, SMA is clinically classified as
the severe type I (Werdnig-Hoffman disease), the moderate type II, and
the mild type III (Kugelberg-Welander disease). The survival motor
neuron (SMN) gene has been established as the disease gene of SMA. The human genome contains two copies of the SMN gene
because of an inverted duplication at 5q13. This phenomenon appears to be human-specific, because all other organisms examined to date have a
single copy of SMN. Deletions or mutations of the telomeric SMN1 gene, which result in reduced SMN protein level, have
been found in the vast majority of SMA patients (4-11).
Although motor neurons seem to be the only known cell type that is
affected in SMA patients, SMN protein is expressed ubiquitously in all
tissues and cell types examined (8, 12, 13). The amino acid sequence of
SMN does not share significant homology with any protein with a known
function; nor does it contain any domains of known function. Although
several lines of evidence have suggested that SMN participates in
several divergent cellular processes, the question of how reduction of
the SMN level leads to motor neuron degeneration remains open. In
addition to its cytoplasmic localization, SMN is found in a novel
subnuclear structure, named gems, which are found in the vicinity of,
and often overlap with, coiled bodies (14). The function of coiled
bodies is unknown, but they contain spliceosomal snRNPs (small nuclear
ribonucleoprotein particles), which function in pre-mRNA splicing,
and components of small nucleolar ribonucleoprotein particles, which
are involved in pre-rRNA processing. This has led to the speculation
that coiled bodies may play some roles in snRNP and small nucleolar
ribonucleoprotein particle metabolism (15). The fact that gems and
coiled bodies are often associated and contain similar sets of proteins
and RNAs suggests that they have related functions. In line with this idea, SMN has been shown to interact with a group of Sm proteins, the
core proteins of snRNPs, and a novel protein, Gemin2 (formerly known as
SIP1), both in vitro and in vivo (14). Injection
of antibodies against either SMN or Gemin2 into Xenopus
oocytes inhibits assembly and import of snRNPs, suggesting that the
SMN-Gemin2 complex performs an important function in snRNP metabolism
(16, 17). A dominant negative mutant of SMN, SMN SMN is evolutionarily conserved throughout eukaryotes, because
homologues of SMN have been identified in many organisms. Genetic studies have shown that SMN is an essential gene in mice,
Caenorhabditis elegans, and Schizosaccharomyces
pombe (28-32). Mice carrying the human centromeric
SMN2 transgene under an SMN null background are
viable and display phenotypes similar to the symptoms of SMA patients,
thereby confirming that SMN is the disease gene of SMA (33,
34). However, little progress has been made from these studies to
elucidate the SMN function.
Homologous recombination occurs at exceptionally high frequencies in
the chicken pre-B cell line DT40, which makes DT40 a useful genetic
system for targeted gene disruption and for studying gene functions
(35). Moreover, disruption of an essential gene is possible in DT40
when the gene product is expressed from a conditional promoter (36). To
gain further insight into the function of SMN, we constructed a DT40
cell line in which the endogenous SMN gene is disrupted, and
SMN protein is produced from an SMN cDNA under the control of the
tetracycline (tet)-repressible promoter. Here we have used this system
to show that SMN is essential for cell viability and that human SMN can
functionally complement for chicken SMN, even though these two proteins
are only ~60% identical in amino acid sequence. This cell line
should provide a powerful system for the characterization of SMN
function and for screening for potential therapeutic drugs for SMA.
Library Screening and Plasmid Constructs--
A chicken
embryonic fibroblast cDNA library (Stratagene) was screened with a
full-length human SMN cDNA radioactively labeled by using a random
labeling kit (Amersham Pharmarcia Biotech). Positive clones were
recovered by in vivo excision according to the
manufacturer's instruction. A chicken genomic library was screened
with a full-length chicken SMN cDNA labeled as above. Positive
clones were mapped by restriction enzyme digestions followed by
Southern blotting. A genomic DNA fragment containing exons 1-5 of
cSMN gene was fully sequenced.
A hygromycin resistance gene under the control of the chicken
Expression vectors VSV-G6 and Hit-60 encoding the envelope glycoprotein
of vesicular stomatitis virus and the Moloney murine leukemia
virus gag-pol protein, respectively, a retroviral vector pMX, and
pMX-enhanced green fluorescence protein are gifts from Dr. Paul Bates
(University of Pennsylvania). To construct the retroviral expression
vectors pMX-cSMN and pMX-huSMN, NcoI-XhoI and
EcoRI-XhoI DNA fragments covering the coding
regions of full-length cSMN and huSMN, respectively, were inserted
between BamHI and XhoI sites of pMX.
Cell Culture and Transfections--
DT40 cells were maintained
in RPMI 1640 medium (Life Technologies, Inc.) containing 10% fetal
bovine serum (Hyclone), 1% chicken serum (Sigma), 2 mM
L-glutamine, and 50 µM 2-mercaptoethanol. DT40 cells were transfected by electroporation as described previously (35). To express cSMN cDNA from a tetracycline-repressible
promoter, DT40 cells were cotransfected with Puro-tTA and 108-cSMN and
selected in medium containing puromycin and histidinol. Concentrations of antibiotics or drugs used in this study are as follows: 1.5 mg/ml
hygromycin B (Calbiochem), 1 mg/ml histidinol (Sigma), 0.5 µg/ml
puromycin (Sigma). Tetracycline hydrochloride was purchased from Sigma
and used as indicated.
Retrovirus Production and Infection--
Transfections of
HEK293T cells were performed by the calcium phosphate method according
to the manufacturer's manual (CLONTECH). To
package retrovirus for infection, HEK293 cells were cotransfected with
10 µg each of VSV-G6, Hit-60, and a pMX vector containing cDNA
of interest. 48 h after transfection, the culture supernatant was
collected, passed through a 0.45-µm filter, supplemented with 10 ng/ml tet, and applied to 1 × 106 S5 cells. 12 h
later, the S5 cells were pelleted and resuspended in RPMI 1640 medium
containing 1 µg/ml tet.
Antibodies and Western Blotting--
Recombinant His-tagged cSMN
protein was used to immunize mice and rabbits to produce polyclonal
antibodies against cSMN. Anti-Gemin2 monoclonal antibody 2E17
and anti-hnRNP A2 monoclonal antibody are described elsewhere
(14).2 The number of cells in
each sample was counted. A pellet of 1 × 106 cells
was lysed in 40 µl of 2× SDS loading buffer. Following a brief
sonication, 20 µl of the lysate was loaded on a 12.5% SDS-PAGE gel.
Western blotting was performed essentially as described (19).
Propidium Iodide Intake Assay--
S5 cells were grown in medium
containing 1 µg/ml tet for 24, 48, 72, or 96 h. Control S5 cells
were maintained in medium containing 10 ng/ml tet. Cells were
collected, washed with 1× phosphate-buffered saline, and resuspended
in 1× phosphate-buffered saline containing 5 µg/ml propidium iodide
at a concentration of 2 × 106 cells per ml.
Immediately thereafter, the samples were analyzed by flow cytometry on
a FACS Scan machine (Becton-Dickinson).
Targeted Disruption of the cSMN Gene--
We cloned cSMN
cDNA by screening a chicken cDNA library with huSMN cDNA as
a probe. The full-length cSMN cDNA encodes a protein of 264 amino
acids, which is 60% identical and 73% similar in sequence to the
huSMN protein (Fig. 1). The cSMN cDNA
was then used to screen a chicken genomic library to obtain genomic DNA fragments covering the entire cSMN locus. A targeting
construct, Hyg-SMN, was made by replacing the entire exon 3 and
part of exon 4 of the cSMN gene with a hygromycin-resistance
gene (Fig. 2A). Following
transfection of wild-type (wt) DT40 cells, screening of over 50 hygromycin-resistant clones by Southern blotting did not yield any
homologous recombinant (data not shown). The intensity of the band on
Southern blots derived from a random integration was the same as that
of the band corresponding to the endogenous locus, indicating that
there is only a single allele of the cSMN gene in DT40
cells. Although the cause of this is not known, we note that a similar
phenomenon, namely that there is a single allele for the tyrosine
kinase gene syk in DT40, has been previously reported
(37).
Given that homologous recombination occurs at a frequency of over 40%
in DT40, the fact that we could not obtain viable clones in which the
single cSMN allele had been disrupted strongly suggests that
cSMN is required for cell viability. To circumvent this problem, we
first established a DT40 cell line, termed C2, which expresses cSMN
protein from a cDNA under the control of a tet-repressible promoter
(38) (see "Materials and Methods"). Addition of 1 µg/ml tet to
the culture medium completely shut off transcription of the cSMN
cDNA. Interestingly, C2 cells maintained in a medium lacking tet
displayed slow growth and cell death, indicating that overexpression of
cSMN is deleterious.3 We
noticed, however, that C2 cells maintained in a medium containing 10 ng/ml tet showed normal growth and expressed high levels of exogenous
cSMN protein (see below). Thus, knockout of the cSMN gene
was carried out in C2 cells cultured in the presence of 10 ng/ml tet.
C2 cells were transfected with linearized Hyg-SMN plasmid DNA by
electroporation and selected in hygromycin-containing medium. Genomic
DNAs were extracted from hygromycin-resistant clones, digested with
XbaI, and subjected to Southern blotting using probe 1 (see
Fig. 2A). Homologous recombinants should contain a longer XbaI fragment (~11 kilobases), because of the
insertion of the hygromycin-resistant gene, and lose the wt
XbaI fragment (~8.5 kilobases; Fig. 2A).
Indeed, several independent clones showed the expected pattern on
Southern blotting and thus are cSMN-knockout cell lines (Fig.
2B). Clone S5 was selected for further investigation and
routinely maintained in a medium containing 10 ng/ml tet. These cells
will be referred to hereafter as control cells unless otherwise indicated.
Depletion of cSMN Protein Results in Cell Death--
To
characterize the expression of cSMN, we produced mouse and rabbit
polyclonal antibodies to it. These antibodies recognized a protein band
of ~38 kDa on Western blots and immunoprecipitated cSMN protein
produced by transcription and translation of the cSMN cDNA in a
rabbit reticulocyte lysate (data not shown). To follow the depletion of
cSMN, cell samples were taken every 8 h, after S5 cells were split
and placed in fresh medium containing 1 µg/ml tet, and analyzed by
Western blotting with an anti-cSMN antibody (Fig.
3A). A lysate of an equivalent
number of cells was loaded on each lane. As indicated above, control S5
cells normally express a high level of cSMN, about 2-3-fold that of wt
DT40 cells (Fig. 3A, first two lanes). Upon
addition of tet, cSMN protein levels decreased, and after 48 h
there was only a trace amount of cSMN protein in S5 cells.
To determine the effect of cSMN depletion on cell growth, we compared
the growth of S5 cells in the presence of either 10 ng/ml or 1 µg/ml
tet. The number of live cells that are round and bright by
phase-contrast microscopy and that are impermeable to the vital stain
trypan blue was counted at the time points indicated (Fig.
3B). A propidium iodide (PI) intake assay was also performed
to measure the extent of cell death (Fig. 3C). Cells that
have a damaged cellular membrane are permeable to PI and are thus
scored as dead. Control S5 cells double every 16 h, considerably
slower than the 10-hour cell cycle of wt DT40 (data not shown). The
slow growth of S5 cells is not caused by overexpression of cSMN but is
rather due to expression of the trans-activator for the tet-repressible
promoter. A similar effect of the tet-off system on cell growth has
been reported previously (36). In the first 48 h, S5 cells
maintained in a medium containing 1 µg/ml tet showed no difference in
growth rate compared with control cells. Between 48 and 72 h, the
number of S5 cells with barely detectable cSMN protein doubled. The
majority of these cells were still alive after 72 h, because there
was only a slight increase in cell death (Fig. 3C), but
massive cell death occurred between 72 and 96 h. PI intake assays
showed that more than 50% of the cells were dead after 96 h (Fig.
3C). Taken together, these results indicate that cSMN is
essential for DT40 cell viability.
Expression of huSMN Rescues Lethality in cSMN-depleted
Cells--
The tet-induced lethality allows us to test whether a given
protein can functionally replace cSMN in a complementation assay. Briefly, S5 cells were transfected with an expression vector containing a cDNA encoding the test protein and then selected in the presence of 1 µg/ml tet. The appearance of surviving cells after 1 week indicates that the test protein can functionally substitute for cSMN
and thus support cell viability. Because of the low efficiency of
transfection of DT40 cells, we used a retroviral infection technique
(39) to transduce cDNAs into these cells. The replication-deficient retrovirus, derived from Moloney murine leukemia virus, was packaged in
HEK293 cells cotransfected with plasmids encoding the envelope glycoprotein of vesicular stomatitis virus and Moloney murine leukemia
virus gag-pol proteins and a retroviral vector containing a
cDNA of the test protein. We found that up to 90% of DT40 cells could be productively infected with a virus containing an enhanced green fluorescence protein cDNA, as determined by the number of cells emitting green fluorescence. As a positive control, S5 cells were
infected with a virus containing the cSMN cDNA. As expected, many
cells survived following tet selection. The growth pattern of the
rescued cells in the presence of 1 µg/ml tet was indistinguishable from that of control S5 cells (data not shown). A Western blot of the
lysate from the rescued cells using an anti-cSMN antibody confirmed
that cSMN was expressed in the presence of 1 µg/ml tet at a level
comparable with that of the control S5 cells (Fig. 4, lane 3). As a negative
control, none of the S5 cells that were infected with a virus
containing the enhanced green fluorescence protein cDNA survived
the tet selection.
Next, we asked whether huSMN can functionally replace cSMN. To this
end, S5 cells were infected with a virus containing the huSMN cDNA
and incubated with 1 µg/ml tet for 1 week. A large number of cells
remained viable after the selection. These cells displayed a growth
rate that was nearly identical to that of the control S5 cells. Lysates
of huSMN-rescued cells were studied by Western blotting using an
anti-cSMN antibody (Fig. 4, lane 4). Previous experiments
have shown that our anti-cSMN antibody cross-reacts with huSMN protein
(data not shown). A protein band migrating slower than cSMN
specifically reacted with the antibody. The same protein band was also
recognized by an anti-huSMN monoclonal antibody, 2B1 (data not shown),
indicating that this band is huSMN. As expected, in the presence
of 1 µg/ml tet, the rescued cells did not express cSMN. Therefore,
expression of huSMN can completely rescue cSMN-depleted cells,
indicating that the function of SMN is highly conserved, if not
identical, between humans and chickens.
Correlation between Cell Growth and Level of cSMN Protein--
The
severity of SMA closely correlates with the degree of reduction of SMN
protein level in SMA patients (12, 13). We wished to determine whether
there is an effect of the level of SMN on S5 cell growth. To this end,
we measured growth rates of S5 cells cultured in medium containing 10, 12, 14, 16, 18, 20, or 100 ng/ml tet (Fig.
5A). To determine cSMN levels
in these cells, lysates of cells grown in the respective media for
72 h were analyzed by Western blotting with an anti-cSMN antibody
(Fig. 5B). In this case, the cSMN level after 72 h of
treatment should reflect the steady state level in cells, because the
protein is depleted after 48 h in the presence of 1 µg/ml tet.
Cells cultured in medium containing 12 and 14 ng/ml tet showed
almost identical growth rates to control cells. Although cSMN levels in
these cells were lower compared with control S5 cells, they were still
equal to or higher than that in wt DT40 cells. A growth defect was seen in cells treated with 16 ng/ml tet, in which the cSMN level dropped to
~30% of that of wt DT40 cells. In the presence of 18 ng/ml tet, cell
growth ceased after 72 h, and at higher concentrations of tet
(e.g. 20 and 100 ng/ml), both growth arrest and cell death were apparent. Therefore, the growth rate of DT40 cells is proportional to the level of cSMN.
Significant Decrease of Gemin2 Protein following cSMN
Depletion--
SMN interacts avidly with Gemin2, and Gemin2
colocalizes with SMN in cells (14). Injection of anti-Gemin2 antibody
into the cytoplasm of Xenopus oocytes inhibits assembly,
maturation, and nuclear import of snRNPs (16). We asked what happens to the amount of Gemin2 following cSMN depletion. The anti-human Gemin2
monoclonal antibody 2E17 recognizes a single band of ~34 kDa on
Western blots of total DT40 cell lysates. In addition, 2E17
coimmunoprecipitates cSMN from total DT40 lysates (data not shown).
Thus, 2E17 cross-reacts with chicken Gemin2. Total lysates of S5 cells
treated with 1 µg/ml tet for 0, 24, 48, or 72 h were resolved by
SDS-PAGE, and a Western blot was performed using anti-cSMN, anti-Gemin2, and, as a control for protein loadings, anti-hnRNP A2
antibodies. As expected, cSMN was not detectable after 48 h (Fig.
6, top panel). Interestingly,
in the same cells a significant decrease of the Gemin2 level was
observed (Fig. 6, middle panel), and it was reduced further
after 72 h. In contrast, the amount of hnRNP A2 remained unchanged
in all samples (Fig. 6, bottom panel). The amounts of hnRNP
C1/C2 proteins and of By generating a cell line with a knockout of the SMN
gene and conditional expression of the SMN protein, we have
demonstrated directly that SMN is required for cell viability. This is
consistent with genetic analysis of the SMN gene in
organisms. Disruption of SMN expression in mice and C. elegans results in early embryonic lethality, which indicates a
requirement of SMN for cell viability in embryos (28, 29). Loss of the
SMN homologue in S. pombe also shows a lethal phenotype
(30-32). Therefore, SMN is required for fundamental cellular processes
that are conserved from fungi to mammals. Although the mice models for
SMA generated recently (33, 34, 40) may be very useful in elucidating
the pathology of SMA, the cell-based genetic system we describe here
provides a setting in which the function of SMN can be studied more
directly, and it offers several unique advantages. First, the SMN level in these cell lines can be modulated precisely and over a broad range,
i.e. from none to 3-4-fold overexpression. Second, the homogeneity of a cell line should greatly facilitate characterization of phenotypes at the molecular level. More importantly, expression of
huSMN protein in cSMN-depleted cells completely rescued the lethal
phenotype, indicating that huSMN performs the same function as cSMN
does, at least at the level of supporting cell viability and
proliferation. Therefore, the function of cSMN defined by characterization of the phenotypes of cSMN-depleted S5 cells directly reflects the function of huSMN in vivo.
The interaction of Gemin2 and SMN has been well characterized (14).
First, Gemin2 directly interacts with SMN in vitro. Second,
Gemin2 can be coimmunoprecipitated with an SMN antibody, and vice
versa. Finally, SMN and Gemin2 are colocalized in cells. Here we
observed a significant decrease of the Gemin2 level concomitant with
cSMN depletion. This confirms and extends to an in vivo
setting the biochemically defined interaction between SMN and Gemin2
and further suggests that components of the SMN-Gemin2 complex are stabilized by forming this complex. The effect of the SMN level on the
stability of Gemin2 is reminiscent of the rapid degradation of the
constituents of TFIID and SAGA complexes upon inactivation of a
single TATA box-binding protein-associated factor subunit (41-45).
TFIID and SAGA are well characterized complexes that function in gene
transcription. It will be interesting to test whether the level of
Gemin2 is also reduced in Type I SMA patients. Additional proteins that
are associated with the SMN-Gemin2 complex have been reported (19, 20),
and it will also be of interest to study what happens to the amounts of
these proteins following SMN depletion.
The molecular basis of SMA is a reduction in the level of SMN protein.
In the most severe type I SMA patients, significant decrease of SMN
protein can be seen in all tissues examined (12, 13). However, motor
neurons appear to be the only cells that are affected. Here we show
that S5 cells that express low levels of cSMN (~30% of the protein
in wt DT40 cells) display slow growth. Considering that DT40 is a pre-B
cell line, this result indicates that cells other than motor neurons
are also dependent on SMN and are sensitive to SMN levels. Overall
growth defects can be seen in SMA mouse models (33, 34). Mice that
express low levels of SMN protein and display SMA symptoms are smaller
than their normal littermates after birth. Thus, it appears that human
cells normally express much more SMN protein than they need, and a
fraction of the SMN pool may actually be sufficient to support normal
growth of cells except for motor neurons. The S5 cell line, whose
growth can be specifically modulated by cSMN levels, provides a
powerful system to search for potential SMA therapies. When maintained to express low levels of cSMN, S5 cells should be useful for high throughput screening for molecules that may be able to increase cell
growth, presumably by enhancing the activity of cSMN, increasing its
production, or slowing its turnover. Furthermore, cSMN-depleted S5
cells should be valuable for high throughput screening for compounds
(if such exist) that can completely substitute for cSMN function. Given the high conservation of the SMN function, the chemical
compounds that we search for will probably exert the same effect on
huSMN and will, therefore, be potential therapeutic drugs for SMA. It
should also be possible to carry out such screening on cSMN-depleted S5
cells whose growth is supported only by (low levels of) huSMN. Such
compounds should also be useful reagents for further understanding of
the normal function of SMN and the pathology of SMA.
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N27, also inhibits snRNP assembly in the cytoplasm (18). Moreover, the nuclear pool of SMN
protein was found to be required for pre-mRNA splicing, probably by
facilitating regeneration or recycling of snRNPs in the nucleus (18).
Recently, two additional proteins, Gemin3 and Gemin4, that are
associated with SMN have been described (19, 20). SMN may also be
involved in regulation of gene expression by interacting with
transcriptional activators (21-23). The ability of SMN to directly
bind RNA, along with its close localization to microtubules in the
cytoplasm and neuronal dendrites and axons, raises a possibility
that SMN is involved in the transport of RNA (24-27).
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-actin
promoter was inserted into cSMN genomic DNA, replacing the entire exon
3 and part of exon 4 of the cSMN gene. To express cSMN
cDNA from a tetracycline-repressible promoter, two plasmid constructs were used, Puro-tTA and 108-cSMN. Puro-tTA containing a
cDNA encoding VP16-tetR chimeric protein (38) under the
control of the chicken
-actin promoter, as well as a puromycin
resistance gene, has been described previously (36). 108-cSMN contains a full-length cSMN cDNA placed downstream of tetO-CMV minimal promoter (38) and a histidinol resistance gene. Details of plasmid constructions are available upon request.
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Fig. 1.
Alignment of huSMN and cSMN. Amino acid
sequences of huSMN and cSMN are aligned by MacVectorTM 6.5. The residues that are conserved between huSMN and cSMN are
boxed. The identical amino acids are shaded dark
and shown as bold, whereas similar amino acids are
shaded light. The GenBankTM accession numbers of
huSMN and cSMN are NM_000344 and AF322650, respectively.
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Fig. 2.
Disruption of the
cSMN gene by homologous recombination.
A, schematic of the cSMN gene locus and gene
targeting strategy. The positions of exons (Ex) 1-4 of the
cSMN gene are indicated as open boxes. The
hygromycin resistance gene (HygroR) is indicated
as a shaded box. The restriction enzyme sites shown are as
follows: H3, HindIII; RI,
EcoRI; SI, SalI;
Sm, SmaI; Xb, XbaI. The
position of the probe (P1) used in Southern blot screening
is indicated. B, C2 cells were transfected with
Hyg-SMN and screened for homologous recombination by Southern
blotting. Genomic DNAs of untransfected DT40 cells (wt) and four
homologous recombinants (S5, S8, m2, and m6) were digested with
XbaI and hybridized with the radioactively labeled probe P1.
The positions of DNA/HindIII markers are indicated.
kb, kilobases.
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Fig. 3.
cSMN is required for DT40 cell
viability. A, S5 cells were grown in medium containing
1 µg/ml tet for the time period indicated on the top.
Lysates of these S5 cells and untransfected DT40 cells (wt) were
resolved by SDS-PAGE and subjected to Western blotting using a rabbit
anti-cSMN polyclonal antibody. The position of cSMN protein is
indicated. B, an equal number of S5 cells were split into
medium containing 10 ng/ml or 1 µg/ml tet. Numbers of live cells were
counted at the time point indicated. The open and
solid boxes indicate the cells cultured in the presence of
10 ng/ml and 1 µg/ml tet, respectively. The growth curve was repeated
at least three times. C, S5 cells were treated with 1 µg/ml tet for 24, 48, 72, or 96 h. These cells and control S5
cells were incubated with 5 µg/ml PI and analyzed by flow cytometry.
The horizontal axis indicates the relative intensity of PI
fluorescence in a single cell. The vertical axis indicates
the cell number. The M1 region represents the live cells that are not
permeable to PI. The M2 region represents the cells that are permeable
to PI and are considered dead. The percentages of M2 region are
indicated.
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Fig. 4.
Expression of huSMN can rescue
cSMN-depleted cells. S5 cells were infected with a retrovirus
containing either cSMN or huSMN cDNA and cultured in the presence
of 1 µg/ml tet for 1 week. Lysates of the viable cells following
infection of cSMN (lane 3) or huSMN (lane 4), the
control S5 cells (lane 2), and untransfected DT40 cells (wt,
lane 1) were resolved by SDS-PAGE and subjected to Western
blotting using a rabbit anti-cSMN polyclonal antibody. The positions of
cSMN and huSMN are indicated.
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Fig. 5.
Growth of S5 cells is proportional to the
level of cSMN. A, after S5 cells were split
into medium containing 10, 12, 14, 16, 18, 20, or 100 ng/ml tet, the
number of live cells was counted every 24 h. The symbols that
indicate different tet concentrations are shown in the panel. The
growth curve was repeated twice. B, S5 cells were treated
with tet of a concentration indicated at the top for 72 h. Lysates of these cells and untransfected DT40 cells (wt) were
resolved by SDS-PAGE and subjected to Western blotting using a rabbit
anti-cSMN polyclonal antibody. The position of cSMN is indicated.
-tubulin were also unchanged in all of these
cells (data not shown). Therefore, depletion of cSMN results in a
specific and proportional reduction of Gemin2 protein in these cells.
This, along with the fact that SMN and Gemin2 associate with each
other, strongly suggests that Gemin2 is stabilized when bound with SMN.
Although we cannot completely rule out the possibility that SMN is
involved in the regulation of the transcription, RNA processing, or
translation of Gemin2 mRNA, the fact that overexpression of cSMN
did not elevate the Gemin2 level (Fig. 6, middle panel,
compare first two lanes) argues against this
possibility.
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Fig. 6.
Reduction of Gemin2 concomitant to cSMN
depletion. S5 cells were grown in medium containing 1 µg/ml tet
for 0, 24, 48, or 72 h. Lysates of these cells and untransfected
DT40 cells (wt) were resolved by SDS-PAGE and subjected to Western
blotting using a rabbit anti-cSMN polyclonal antibody (top),
an anti-Gemin2 monoclonal antibody 2E17 (middle), or an
anti-hnRNP A2 monoclonal antibody (bottom). The positions of
cSMN, Gemin2, and hnRNP A2 are indicated.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Linda Abel and Robert Perkinson for producing mouse anti-cSMN polysera, Lili Wan for sharing the anti-hnRNP A2 monoclonal antibody, and members of our laboratory for helpful discussions. We thank Drs. Zissimos Morelatos, Westley Freisen, Livio Pellizzoni, and Amelie Gubitz for comments on this manuscript. We are grateful to Dr. Paul Bates for providing us with the retroviral expression system.
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FOOTNOTES |
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* This work was supported by a grant from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF322650.
An investigator of the Howard Hughes Medical Institute. To whom
correspondence should be addressed. Tel.: 215-898-0398; Fax: 215-573-2000; E-mail: gdreyfuss@hhmi.upenn.edu.
Published, JBC Papers in Press, December 19, 2000, DOI 10.1074/jbc.M009162200
2 L. Wan and G. Dreyfuss, unpublished data.
3 J. Wang and G. Dreyfuss, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: SMA, spinal muscular atrophy; SMN, survival motor neuron; snRNP, small nuclear ribonucleoprotein particle; tet, tetracycline; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; cSMN, chicken SMN; huSMN, human SMN; wt, wild-type; PI, propidium iodide.
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REFERENCES |
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