A degron created by SMN2 exon
7 skipping is a principal
contributor to spinal muscular
Sungchan Cho and Gideon Dreyfuss1
Howard Hughes Medical Institute and Department of
Biochemistry and Biophysics, University of Pennsylvania
School of Medicine, Philadelphia, Pennsylvania 19104, USA
Spinal muscular atrophy (SMA) is caused by homozygous
survival of motor neurons 1 (SMN1) gene deletions,
leaving a duplicate gene, SMN2, as the sole source of
SMN protein. However, most of the mRNA produced
from SMN2 pre-mRNA is exon 7-skipped (~80%), re-
sulting in a highly unstable and almost undetectable
protein (SMND7). We show that this splicing defect cre-
ates a potent degradation signal (degron; SMND7-DEG) at
SMND7’s C-terminal 15 amino acids. The S270A muta-
explain a key aspect of the SMA disease mechanism, and
suggest new treatment approaches based on interference
with SMND7-DEG activity.
Supplemental material is available at http://www.genesdev.org.
Received November 11, 2009; revised version accepted
January 13, 2010.
Spinal muscular atrophy (SMA) is a common and often
fatal motor neuron degenerative disease, and a leading
genetic cause of infant mortality (Talbot and Davies 2001;
Wirth et al. 2006a; Burnett et al. 2009a). SMA severity
corresponds to the degree of functional survival of motor
neurons (SMN) protein deficiency. SMN is a ubiquitously
expressed protein that plays a critical role in RNA
metabolism, and is essential for viability of all cells in
eukaryotes (Yong et al. 2004; Neuenkirchen et al. 2008).
As part of a large multiprotein complex, the SMN com-
plex, SMN functions in the biogenesis of small nuclear
ribonucleoproteins (snRNPs), the major subunits of the
spliceosome (Fischer et al. 1997; Liu et al. 1997; Meister
et al. 2001; Pellizzoni et al. 2002). Although SMN de-
ficiency manifests itself as a motor neuron disease, its
molecular consequences are evident as profound disrup-
tions in RNA metabolism in all tissues tested in an SMA
mouse model (Gabanella et al. 2007; Zhang et al. 2008).
There are two SMN genes in humans, SMN1 and SMN2,
both encoding the same ORF. The vast majority of SMA
patients have homozygous SMN1 deletions and are sus-
tained by one or more copies of SMN2. However, due to
a C/T substitution at position 6 of exon 7 that does not
change the encoded amino acid, the splicing of the SMN2
pre-mRNA incurs frequent (;80%) exon 7 skipping. This
produces an SMN protein (SMND7) that lacks the normal
C-terminal 16 amino acids and acquires instead four
amino acids, EMLA, encoded by exon 8 (Le et al. 2005).
Thus, SMN1 deletions expose the splicing defect of SMN2
and its ineffectiveness in producing full-length normal
SMN protein (Wirth et al. 2006a; Cooper et al. 2009).
Biochemical experiments in vitro suggested that SMND7
is not fully functional compared with normal SMN pro-
tein, including a diminished oligomerization and binding
to protein substrates such as the snRNP Sm proteins
(Lorson et al. 1998; Pellizzoni et al. 1999). However, as
SMND7 is extremely unstable and is generally undetect-
able, a definitive measure of its functional deficit in cells
has not been possible. Nevertheless, increased SMN2
copy number correlates with a milder clinical phenotype
in SMA patients (Wirth et al. 2006b). For example, severe
SMA (type I) patients typically have one or two SMN2
copies, intermediate severity SMA (type II) patients
usually have three SMN2 copies, and patients with mild
SMA (type III) mostly have three or four SMN2 copies
(Feldkotter et al. 2002; Cusco et al. 2006). Furthermore,
studies in cells (Wang and Dreyfuss 2001b) suggested—
and experiments in SMN-deficient mice demonstrated—
that expression of an increasing copy number of SMND7
cDNA transgenes proportionately lessens SMA severity
(Le et al. 2005). This suggests that even a modest SMND7
increase is beneficial in SMA. With this in mind, our
experiments here were designed to determine the cause
of SMND7 instability.
Results and Discussion
We first established a reporter system that recapitulates
the differential stability of full-length SMN and SMND7
and allows quantitative assessment of SMND7’s insta-
bility determinants. Luciferase (Luc) reporter proteins
consisting of normal SMN or SMND7 fused to the C
terminus of Luc were produced by transfection of the
corresponding cDNA constructs in 293T cells (Fig. 1).
Forty-eight hours after transfection, cells were treated
with the protein synthesis inhibitor cycloheximide
(CHX), and Luc activity was measured at time intervals
of up to 10 h. Consistent with previous reports (Lorson
and Androphy 2000), SMN has a half-life of >8 h, whereas
SMND7 has a half-life of ;3 h. After 10 h of CHX chase,
there was three times more SMN than SMND7. Several
constructs were prepared to determine the role of the
C-terminal sequence of SMND7 in this protein’s instabil-
ity. Deletion of the C-terminal EMLA from SMND7
(SMND7DEMLA) increased the half-life of SMND7 by
twofold (Fig. 1B), and a further deletion of the YG box
(SMND7DYG), a conserved tyrosine/glycine-rich motif in
divergent SMNs (Talbot et al. 1997) that is essential for
SMN oligomerization (Pellizzoni et al. 1999), also had the
same effect. These results suggest that EMLA and the YG
box are major contributors to SMND7’s instability. Im-
portantly, YG + EMLA alone was sufficient to cause
dramatic instability of Luc that is similar to that of
SMND7. Neither YG nor EMLA alone was sufficient for
full destabilization activity (Fig. 1B). N-terminal deletions
[Keywords: Survival of motor neurons (SMN); spinal muscular atrophy
(SMA); motor neuron degenerative disease; protein degradation signal
(degron); protein stability; pre-mRNA splicing]
E-MAIL firstname.lastname@example.org; FAX (215) 573-2000.
Articleis online at http://www.genesdev.org/cgi/doi/10.1101/gad.1884910.
438GENES & DEVELOPMENT 24:438–442 ? 2010 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/10; www.genesdev.org
in the YG box decreased the destabilizing activity of YG +
EMLA (data not shown). These data indicate that YG +
EMLA, corresponding to SMND7 amino acids 268–282, is
the minimal sequence required for full SMND7 destabi-
lization, and is both necessary and sufficient to trigger
rapid degradation of a heterologous protein.
As a further test of this conclusion, we fused YG +
EMLA to another reporter, GFP, and expressed this pro-
tein as well as GFP as a control in 293T cells. The GFP
signal from GFP-YG + EMLA, as determined by Western
blots, showed a gradual decrease after treatment with
CHX (Fig. 2A). GFP-YG + EMLA protein decreased faster
than GFP-NS (nonspecific sequence), and the half-life of
GFP-YG + EMLA was about half that of GFP-NS. These
results suggest that YG + EMLA functions as a protein
degradation sequence. For comparison, we tested the de-
stabilizing activity on the same reporter of YG + EMLA
and an optimized PEST, a potent and well-characterized
protein destabilizing signal (Li et al. 1998). YG + EMLA
had a similar effect to that of the genetically improved
PEST (Fig. 2B). Furthermore, SMN Exon6 + EMLA, which
is the same size as the 41-amino-acid PEST sequence,
conferred similar instability. As this optimized PEST
sequence has about half the half-life of the natural one
(Li et al. 1998), YG + EMLA could be estimated to have
similar or stronger destabilization activity than that of
the natural PEST sequence, and Exon6 + EMLA is about
twice as strong. These data demonstrate that YG + EMLA
is a highly potent and transferable protein degradation
signal (degron), which we term SMND7-DEG, for SMND7
degron. Addition of five amino acids to the C-terminal
end of EMLA (SMND7 + 5aa) caused SMND7 stabiliza-
tion, indicating that SMND7-DEG must be exposed at the
C terminus of the protein for activity (Fig. 2B). This is
consistent with the observation that several additional
amino acids, which can be effected by aminoglycoside-
forced translational read-through, enhanced SMND7 sta-
bility and functionality (Mattis et al. 2008; Heier and
SMN was shown previously to be degraded by the
proteasome (Chang et al. 2004; Burnett et al. 2009b). To
determine if SMND7 is also degraded by this system, cells
expressing Luc-SMND7 were treated with proteasome
inhibitors (MG132 and Lactacystin) for 5 h in the pres-
ence of CHX. CHX treatment alone resulted in a 60%
decrease in signal, but a much smaller decrease was seen
in the presence of proteasome inhibitors MG132 and
Lactacystin (Supplemental Fig. 1). Inhibitors of other
proteolytic activities—such as lysosomal proteases, auto-
phagy, and calpain (NH4Cl, 3-methyladenine, and cal-
peptin, respectively)—had no effect. These data dem-
onstrate that SMND7 is degraded by the proteasome.
To identify specific residues in the SMND7-DEG that
are important for its activity, we performed mutagenesis
of the YG box, converting every second residue to alanine
in the context of full-length SMND7, and determined the
half-life of each in 293Tcells. Of seven mutations tested,
S270A produced the most striking effect, reversing the
destabilizing activity of the SMND7-DEG (Fig. 3A). To
confirm that S270A stabilizes SMND7, HA-tagged pro-
teins SMN, SMND7, and SMND7S270Awere expressed in
293Tcells for 24 h, and then treated with the proteasome
sequence in SMND7. (A) Schematic diagram of Luc-fused SMN and
a series of deletion constructs used for quantitative measurement of
protein stability. Shown are SMN exon structures. YG box denotes
the tyrosine/glycine (YG)-rich sequences in exon 6 of SMN. The
EMLA sequence encoded by exon 8 is depicted by the red box at the
C-terminal end of SMND7. (B) 293T cells were transfected with
plasmids expressing Luc-SMN, Luc-SMND7, and the indicated de-
letion constructs. Forty-eight hours after transfection, the cells were
treated with CHX (0.1 mg/mL) for various times as indicated, and
then assayed for luciferase activity. Luc activity at each time point
was calculated by comparison with those at time 0, which was set to
100%. Fifty percent activity is indicated by the gray dotted line.
Error bars represent standard deviation (SD) from three independent
Delineation of YG + EMLA as a protein destabilization
protein destabilizing signal (degron). (A) Plasmids expressing GFP-
YG + EMLA or GFP-NS (nonspecific sequence) were transfected into
293T cells. Twenty-four hours after transfection, the cells were
treated with CHX (0.1 mg/mL) for various times as indicated. GFP
fusion proteins were detected by Western blot using anti-GFP
antibody, and Magoh was used as a loading control. (B) Comparison
of YG + EMLA and Exon6 + EMLA of SMND7 with an optimized
protein-destabilizing element (optiPEST). Shown also are Luc-SMN
and Luc-SMND7 containing an additional five amino acids at the
C-terminal end (SMND7 + 5aa). Luc activities were measured as in
Figure 1. Error bars represent SDs from three independent experi-
The C terminus of SMND7, YG + EMLA, is a strong
SMND7 degron contributes to SMA severity
GENES & DEVELOPMENT 439
inhibitor MG132 for 16 h. The levels of the tagged SMN
proteins were then monitored by Western blots using
anti-HA antibody (Fig. 3B). As expected, the amount of
SMND7 without MG132 treatment was much lower than
that of SMN. However, the amount of SMND7S270Awas
similar to that of normal SMN, indicating an almost
complete restoration of stability by S270A mutation.
MG132 caused a dramatic increase in the amount of
SMND7 (;3.4-fold), but only a moderate effect on SMN
and SMND7S270A(Fig. 3C). Therefore, the S270A muta-
tion limits the proteosome degradation of SMND7 and
increases its stability very significantly. We further tested
the effect of S270A in the context of SMND7-DEG alone.
S270A mutation strongly increased the stability of Luc-
YG + EMLA to a level similar to that of SMN (Fig. 3D).
These data indicate that the enhancement of stability of
SMND7 by the S270A mutation occurs through SMND7-
To determine whether SMND7S270Ais a functional
SMN protein, we used a previously established cell
system, the S5 cell line, to ask if it could rescue the
viability of SMN-depleted cells. S5 is derived from
chicken DT40 cells in which the endogenous chicken
SMN gene is disrupted by homologous recombination and
SMN protein is expressed exogenously from a cDNA
under a tetracycline-repressible promoter (Wang and
Dreyfuss 2001a). Upon depletion of chicken SMN
(cSMN), S5 cell growth arrests at 72 h and cell death
occurs. It is therefore useful to assess the physiological
functionality of SMN mutants in this cell system by
monitoring cell viability after turning off cSMN cDNA
expression and simultaneously expressing exogenous
SMN mutants of interest. To do so, we constructed
recombinant retroviruses expressing SMN, SMND7, or
SMND7S270Aand transduced S5 cells. One week after
repression of cSMN expression by tetracycline (1 mg/mL),
there was a very clear difference in viable cell number
among three samples (Fig. 4A,B). As expected (Wang and
Dreyfuss 2001b), while SMN rescued the viability of S5
cells, SMND7 did not. Importantly, SMND7S270Aalso
rescued S5 cells to a similar extent as SMN. The two
rescued cell lines expressed a similar level of SMN pro-
tein (Fig. 4C). Since the deficiency in functional SMN
protein is correlated directly with snRNP assembly de-
fects in cells of SMA patients (Wan et al. 2005), we next
examined whether SMND7S270Ais active in snRNP
assembly as a further measure of functionality. Extracts
alanine as indicated. All constructs had Luc fusions, and Luc activity was assayed as in Figure 1. (B) HA-tagged SMN, SMND7, and SMND7S270A
were expressed in 293T cells for 24 h, and then cells were treated with DMSO (D) or 10 mM MG132 (MG) for 16 h. Fusion proteins were
monitored by Western blot using an anti-HA tag antibody, and Magoh was used as a loading control. (C) HA-tagged proteins in B were quantified
and compared with HA-SMN without MG132 treatment, which was set to 100%. The fold change of each fusion protein amount upon MG132
treatment is indicated in red above the column. (D) All constructs had Luc fusions, and Luc activity was assayed as in Figure 1. Error bars
represent SDs from three independent experiments.
S270 is critical for the activity of the SMND7-DEG through YG + EMLA. (A) Seven residues in the YG box were each mutated to
tional in snRNP assembly. (A) S5 cells were cultured in the presence
of tetracycline (1 mg/mL) to deplete endogenous SMN, and were in-
fected with retroviruses expressing SMN, SMND7, or SMND7S270A.
One week after tetracycline addition, cells were stained with Trypan
blue and visualized by DIC microscopy. (B) Cell growth as in A was
measured by monitoring the number of live cells at the indicated
time points following tetracycline addition. (C) Western blots of
SMN protein in rescued cells (10 d after tetracycline addition).
(D) Cytoplasmic extracts from rescued cells were assayed for
snRNP assembly on U4 snRNA in vitro, using U4DSm RNA as a
SMND7S270Arescues SMN-deficient cells and is func-
Cho and Dreyfuss
440GENES & DEVELOPMENT
from cells expressing SMN and SMND7S270Awere pre-
pared, and their snRNP assembly activity was de-
termined by measuring the amount of Sm protein cores
that form on biotinylated snRNA substrate captured on
streptavidin beads (Wan et al. 2005). Sm cores are the
major constituentsof snRNPs whose assembly on snRNAs
depends on the SMN complex. As shown in Figure 4D,
both cell lines showed similar activity. These data in-
dicate that SMND7S270Ais a functional protein similar to
normal SMN in S5 cells. We conclude that the instability
of SMND7 conferred by SMND7-DEG is a principal
contributor to the deleterious phenotype of exon 7
skipping, and that S270A substitution in SMND7 abro-
gates the degron activity, thereby restoring the function
Several diverse classes of degrons that target proteins
to various degradation pathways have been described.
Most noted are N degrons comprised of destabilizing
N-terminal residues, C-terminal determinants containing
relatively unstructured hydrophobic residues, and phos-
pho-degrons that are modulated by the phosphorylation
status of their serine/threonine residues in response to cell
signaling (Parsell et al. 1990; Ravid and Hochstrasser
2008). The short-lived tumor suppressor protein PTEN’s
stability depends on a 50-amino-acid C-terminal tail that
is phosphorylated at specific serine/threonine residues
(Vazquez et al. 2000). Interestingly, while many of the
residues of the SMND7-DEG could be substituted by
alanines without loss of degron function, S270 is critical
for the destabilizing function. It is therefore possible that
S270 is phosphorylated, and that this regulates the
SMND7-DEG activity. However, phosphorylation site
analysis by NetPhos did not reveal strong candidate
kinases for it. SMND7-DEG has no obvious sequence
similarity with the known degrons and, thus, represents
a novel protein-destabilizing element. Protein database
searches did not identify other known proteins contain-
ing sequences highly similar to SMND7-DEG.
The reduced oligomerization efficiency of SMND7 has
been suggested recently to account for its instability
(Burnett et al. 2009b). Indeed, intermolecular SMN oxi-
dative cross-linking provided direct evidence that SMN is
oligomeric in cells (Wan et al. 2008). Oligomerization is
likely to be important for SMN function, and also to
contribute to its stability. However, although SMN olig-
omerization correlated with its stability, this did not ex-
plain the intrinsic instability of SMND7. Our findings
show that attachment of SMND7-DEG to monomeric
protein reporters (Luc and GFP) triggered their rapid
degradation, indicating that lack of oligomerization is
not the major cause of SMND7’s instability. Loss of
oligomerization capacity and other possible deficits as
a result of deletion of the peptide encoded by exon 7 may
result in an SMN protein that is functionally suboptimal.
However, the detrimental effect of exon 7 skipping does
not arise primarily from deletion of a functionally essen-
tial domain, but from the creation of a positively act-
ing and potent degron that causes severe deficiency of
Given the ability of S270A mutation to restore
SMND7’s stability and complement SMN loss of func-
tion, it is reasonable to predict that polymorphisms that
inactivate SMND7-DEG, such as at S270, would result in
a milder SMA phenotype than the genotype predicts
based on SMN2 copy number in SMN1-deleted individ-
uals. Our finding with SMND7S270A
SMND7 is a functional SMN protein, and that its stabi-
lization could prevent or lessen SMA severity. We suggest
that interfering with SMND7-DEG activity could be an
effective approach for mitigating its deficiency as a poten-
tial treatment for SMA. Although the inhibitor studies
suggest that the degradation of SMND7 likely occurs in
the proteasome, general inhibition of proteasome activity
would be very toxic, particularly in the long-term treat-
ment that SMA would be expected to require. A targeted
inhibition of the factors that mediate the SMND7-DEG-
dependent degradation should provide a more specific
therapeutic approach, and their identification will be of
great interest for SMA therapy.
SMA is thus the result of a fateful chain of events.
Homozygous SMN1 deletion is a cause of SMA only
because it exposes the splicing defect of SMN2. We argue
that the splicing defect in SMN2 causes SMN deficiency
because it fortuitously creates a degron. The degron is
a key to SMA, as it is the most direct cause of SMN
deficiency, which then results in major perturbations in
Materials and methods
Plasmid construction and generation of mutations
To construct plasmids expressing Luc-fused proteins, the Luc gene was
cloned into pcDNA3.1 vector at HindIII/KpnI sites, and then DNA frag-
ments encoding full-length wild-type human SMN, SMND7, several dele-
tion mutants of SMND7, and optiPEST were inserted into the KpnI/XhoI
sites. SMND7 mutants with a single amino acid change were generated by
mutating residues in YG + EMLA to alanine by QuickChange site-directed
mutagenesis kit (Stratagene). Plasmid expressing GFP-YG + EMLA was
constructed by inserting a DNA fragment encoding YG + EMLA into
pEGFP vector (Clontech) at KpnI/BamHI sites. Plasmids expressing HA-
SMNs were constructed by inserting DNA fragments encoding HA-tagged
SMN, SMND7, and SMND7S270Ainto the BamHI/XhoI sites of pcDNA3
vector. To generate retroviral plasmids to express SMNs in S5 cells, DNA
fragments encoding SMN, SMND7, and SMND7S270Awere cloned into the
EcoRI/XhoI sites of pMX vector as described (Wang and Dreyfuss 2001a).
Assays for protein stability
Luc- and GFP-based assays were performed as described in the legends for
Figures 1 and 2A, respectively. Luc activities were measured using One-
Glo reagent (Promega).
Rescue of S5 cell viability
S5 cells were maintained and infected with retroviruses expressing SMN,
SMND7, and SMND7S270Aas described (Wang and Dreyfuss 2001a).
SMN complex activity assay
Cytoplasmic extracts from rescued S5 cells were prepared and assayed for
snRNP assembly in vitro as described (Wan et al. 2005).
Mouse monoclonal antibodies anti-SMN (62E7) and anti-Magoh (18G12)
were used as described previously (Wan et al. 2005). Rabbit polyclonal
antibodies anti-HA (Santa Cruz Biotechnologies) and anti-GFP (Santa
Cruz Biotechnologies) were used as recommended by the manufacturer.
We thank the members of our laboratory, especially Dr. Lili Wan, for
helpful discussions and comments on this manuscript. This work was
SMND7 degron contributes to SMA severity
GENES & DEVELOPMENT441
supported by the Association Franc xaise Contre les Myopathies (AFM).
G.D. is an Investigator of the Howard Hughes Medical Institute.
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