Advanced Review

SMN in spinal muscular atrophy

and snRNP biogenesis
Tristan H. Coady† and Christian L. Lorson∗

Ribonucleoprotein (RNP) complexes function in nearly every facet of cellular

activity. The spliceosome is an essential RNP that accurately identifies introns
and catalytically removes the intervening sequences, providing exquisite control
of spatial, temporal, and developmental gene expressions. U-snRNPs are the
building blocks for the spliceosome. A significant amount of insight into the
molecular assembly of these essential particles has recently come from a seemingly
unexpected area of research: neurodegeneration. Survival motor neuron (SMN)
performs an essential role in the maturation of snRNPs, while the homozygous
loss of SMN1 results in the development of spinal muscular atrophy (SMA), a
devastating neurodegenerative disease. In this review, the function of SMN is
examined within the context of snRNP biogenesis and evidence is examined which
suggests that the SMN functional defects in snRNP biogenesis may account for the
motor neuron pathology observed in SMA.  2011 John Wiley & Sons, Ltd. WIREs RNA
2011 2 546–564 DOI: 10.1002/wrna.76

SURVIVAL MOTOR NEURON
S urvival motor neuron (SMN) is a 38 kDa
protein, encoded by nine exons (Figure 1), that is
ubiquitously expressed throughout development and
has been identified as a protein-binding partner for
nearly 50 cellular and viral proteins.1,2 While the
SMN expression can be detected in all tissues, levels
are not equivalent.3 SMN has been implicated in a
variety of cellular activities, including transcription,
RNA stability, apoptosis, pre-mRNA splicing, stress
responses, axonal RNA trafficking, and snRNP
biogenesis.4 In the early analysis of the SMN protein, it
was identified in punctate nuclear structures originally
referred to as Gemini-of-coiled-bodies or gems5,6
(Figure 2). These nuclear structures are distinct from
splicing speckles and other nuclear structures such
as PML bodies, however, gems—as the Gemini-
derived moniker suggests—are frequently immediately
adjacent to or overlapping Cajal bodies.5–10 Gems are
enriched in a variety of cellular factors, including
those involved in RNA processing and transcription
†
Current address: Department of Biological Sciences, Columbia
University, New York, NY, USA
∗ Correspondence to: lorsonc@missouri.edu
Department of Veterinary Pathobiology, Bond Life Sciences Center,
University of Missouri, Columbia, MO, USA
DOI: 10.1002/wrna.76
regulation, however, they are not active sites of pre-
mRNA processing.11 SMN is also diffusely present in
the cytoplasm where it performs its most thoroughly
described biochemical function: snRNP assembly and
maturation.12,13 SMNs role in snRNP biogenesis and
its relation to SMA will be the focus of this review.
SMN AND SPINAL MUSCULAR
ATROPHY
Spinal muscular atrophy (SMA) is a leading genetic
cause of infantile death worldwide and is the second
most common autosomal recessive disorder.14,15 SMA
is a neurodegenerative disease that occurs in approx-
imately 1:6000 live births and has a carrier frequency
between 1:30 and 1:40.16,17 The overwhelming major-
ity of the clinical manifestations in SMA are due to
the progressive loss of α motor neurons within the
anterior horns of the spinal cord. Loss of the motor
neurons and disruption of the motor unit results in
the atrophy of proximal voluntary muscles, eventually
progressing to the point where the trunk muscles are
compromised. Respiratory complications are a major
cause of mortality. There are four clinical forms of
the disease, based upon the age of disease onset,
the severity of the symptoms, and the achievement
of defined physical milestones.18–21 At the molecular
level, all SMA patients lack SMN1; however, the copy

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 WIREs RNA SMN in spinal muscular atrophy and snRNP biogenesis

Cytoplasmic
localization/
Nucleic acid Tudor domain Y/G stability
binding Sm-fold Poly-proline box

AA 28 52 92 159 210 242 279 294

Exon 1 2a 2b 3 4 5 6 7

Oligomerization Sm core, Profilin Oligomerization

Coilin
Gemin2 fibrillarin
GAR-1

FIGURE 1 | Survival motor neuron (SMN) protein subdomains. The amino acid number corresponding to the exon peptides and the size of the
SMN protein is indicated above the red exon boxes. The SMN is 294 amino acids and runs at 38 kDa on Western blots. The relative sizes of the exons
are diagrammed with the exon number below. Identified protein subdomains and associated functions are indicated. Below is an abbreviated list of
proteins that interact with the SMN along with the approximate region of interaction.

can never stand without assistance. Typically, the dis-

FIGURE 2 | Intracellular localization of the survival motor neuron
(SMN). An anti-SMN monoclonal antibody (4B7) was used to
specifically detect SMN (green) in the HeLa cells, while DAPI stained the
nucleus. The SMN is present within discrete nuclear foci, often referred
to as gems. These gems have been used as a surrogate marker for the
overall SMN concentration as the gem numbers frequently correspond
to the total intracellular SMN levels.
number of a nearly identical copy gene called SMN2
is a critical disease modifier as less severe forms of
the disease are associated with increased SMN2 gene
copies (Figure 3). Type I SMA or Werdnig–Hoffmann
disease, is the most severe form and is by far the most
common, comprising nearly 60% of all newly diag-
nosed cases. While the life expectancy has improved
for SMA I patients in the developed world, it is due in
large part to supportive care. In the absence of aggres-
sive intervention, life expectancy is less than 2 years.22
Type II SMA is less severe, where patients can sit, but
ease presents after 2 years of age and patients have
a significantly longer life span compared to Type I
patients.21,23,24 Type III SMA or Kugelberg–Welander
disease, is often referred to as the juvenile form of SMA
because symptoms typically develop later in life and
all patients can walk unassisted at some point during
their lives.24 Life span is normal for Type III individu-
als. Type IV SMA, often referred to as the adult form,
is the least severe form and onset typically occurs in
patients older than 30 years.2 Although these defini-
tions are useful, SMA is an extremely heterogeneous
disease. There are many cases that do not fall cleanly
into one category; conversely, there is additional strat-
ification within these classifications, used for clinical
management as well as clinical trials development.
Full-length SMN is actually produced by two
nearly identical genes, SMN1 and SMN2.1 These
genes are located on the long arm of chromo-
some 5 and are present within two relatively large
genomic repeats consisting of approximately 500 kb
(Figure 4). Several additional genes are present within
the repeats, although they are not involved in the
SMA development.1,25 The repeats lie in a head-to-
head orientation and are regulated by nearly identical
promoter elements. SMN1 expresses high levels of
full-length SMN, while SMN2 produces low levels of
full-length SMN transcript and an abundance of an
alternatively spliced isoform that lacks the final coding
exon, Exon 7. Although RT-PCR can detect the pres-
ence of other alternatively spliced isoforms lacking
Exons 5, 7 or both 5 and 7,26 these are only produced
at very low levels and it is clear that the critical differ-
ence between SMN1 and SMN2 is in the processing

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 Advanced Review wires.wiley.com/rna

Unaffected Embryonic lethal Type I SMA

SMN2 SMN1 SMN2 SMN1 SMN2 SMN1

SMN2 SMN1 SMN2 SMN1 SMN2 SMN1

Type II SMA Type III SMA Unaffected carrier

SMN2 SMN1 SMN2 SMN2 SMN2 SMN2

SMN2 SMN2 SMN2 SMN2 SMN2 SMN2 SMN2

FIGURE 3 | SMN2 is a critical modifier of disease severity. The genomic organization of a typical unaffected individual contains two SMN1 and
two SMN2 genes, one on each chromosome (5q). No individuals have been identified that lacked both SMN1 and SMN2, presumably because this
would result in early embryonic lethality. As the SMN2 gene copy number increases in the SMN1-null spinal muscular atrophy (SMA) patients, the
disease severity tends to decrease. This is likely due to the low, residual level of full-length SMN produced by each SMN2, approximately 10% per
SMN2 copy. High SMN2 numbers have been observed in SMN1-null individuals who are phenotypically normal, although in genetic terms are defined
as having SMA.

5q11.2-13.3 its total absence.30–32 This is why no SMA patients

500 kb 500 kb
GTF2H2T NAIPψ SMN2 SERF1C SERF1T SMN1 NAIP GTF2H2T
Sp1 Ets
+1
Caa
g G
1 2a 2b 3 4 5 6 7 8
a gg A sistent with the majority of SMA cases in which
T
FIGURE 4 | Genomic organization of the survival motor neuron
(SMN) locus. SMN1 and SMN2 are present on a single region of the
same chromosome: 5q11.2-13.3. An evolutionarily recent genetic
duplication of approximately 500 kb resulted in the duplication and
inversion of the SMN and several flanking genes, including SERF1,
NAIP, and GTF2H2. Initially, genes other than SMN were implicated in
the spinal muscular atrophy (SMA) development because larger
deletions frequently excised SMN and additional sequences. However,
the detection of intragenic mutations within the SMN conclusively
demonstrated that SMN was the only SMA-determining gene. SMN1
and SMN2 are nearly identical and the regulatory mechanisms
governing their promoters are essentially unchanged (Sp1, Ets sites, and
many more). Methylation and histone regulation appear critical for the
SMN expression. Five silent, nonpolymorphic nucleotide differences are
indicated at the 3 end of SMN1 (SMN1-derived sequences are on the
top; SMN2 sequences are at the bottom). The critical difference is within
Exon 7 (a C–T transition). In the full-length transcript, Exon 8 is entirely
noncoding, while in the SMN7 transcript, four additional amino acids
are derived from the 5 end of Exon 8.
of SMN Exon 7.27,28 Interestingly, only the loss of
SMN1 results in SMA development whereas the loss of
SMN2 has no clinical consequence.29 It is important to
stress that complete loss of SMN is a lethal condition
and that SMA is caused by low levels of SMN—not
have been identified, who are null for both SMN1 and
SMN2. While humans are the only species with SMN1
and SMN2 genes,33 work performed in mice is consis-
tent with the notion that low levels, not the complete
absence, of SMN results in SMA.32,34 The knockout of
the murine SMN gene, which is functionally equivalent
to SMN1, results in pre-implantation lethality, while
introduction of the human SMN2 in the SMN-null
background confers viability.32 This work is con-
increasing SMN2 copy numbers are associated with
less severe forms of disease14 (Figure 3). It is impor-
tant to stress that the truncated product produced by
SMN2, often referred to as the SMN7 protein, is not
a dominant-negative factor and does retain some resid-
ual activity compared to the full-length SMN.35–37
SMN Pre-mRNA Splicing
While SMN1 and SMN2 encode identical amino acid
sequences, the majority of SMN2-derived transcripts
are alternatively spliced and lack Exon 7, encoding the
SMN7 protein1,26 (Figure 5). This protein is unable
to form complexes with most of the typical SMN-
associated protein and RNAs and thus is unstable and
is rapidly degraded.38,39 A number of recent reviews
have focused upon the dynamic interplay between
the complex regulatory factors governing SMN Exon
7 splicing; therefore, only a brief description of this
regulatory aspect will be included.40–43
Pre-mRNA splicing of SMN Exon 7 is regulated
by a variety of cis- and trans-acting factors.2,42,44,45
Some elements are essential for Exon 7’s inclusion
for either SMN1 or SMN2, such as the hTraβ1-
binding site located within the central region of the 54

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 WIREs RNA SMN in spinal muscular atrophy and snRNP biogenesis

SMN1
hnRNP-G
U2 SRp30c
snRNP
hnRNP-Q
U2AF35 SF2/ASF hTra2-β1 70K
U2AF65 U1 snRNP
Exon 6 CAGACAA Exon 8

SE1 SE2 SE3 Element 2

100% 100%
SMN exon 7 region

U2
SMN2 snRNP
U2AF35
U2AF65 70K
U1 snRNP
Sam68

hnRNP-A1
PTB/FUSE hnRNP-A1 hnRNP-A1 hnRNP-A1
Exon 6 TAGACAA Exon 8
G
EXTINCT ISS-N1

10% 90% 10%

FIGURE 5 | Pre-mRNA splicing regulation of survival motor neuron (SMN) genes. SMN1 exclusively produces the full-length form of the protein.
A number of cis - and trans -regulatory elements facilitate SMN Exon 7 inclusion. Positively acting factors are shown in the top panel, including various
SR, SR-like, and heterogeneous nuclear ribonucleoprotein (hnRNP) proteins (green), as well as putative splice enhancers (SE1–3) and Element 2. The
short sequence motif ‘CAGACAA’ represents a high-affinity SF2/ASF-binding motif that is bound by SF2 only in the SMN1 context. The first ‘C’
position (+6) is altered in SMN2 to a ‘T’. The general splicing machinery is indicated (blue). The bottom panel represents the regulatory mechanisms
governing SMN2 pre-mRNA splicing. The SF2/ASF-binding motif has been severely impaired by the introduction of a ‘T’ at the +6 position:
‘TAGACAA’. Additionally, this site is now bound efficiently by hnRNP-A1 and is within an additionally regulatory element referred to as EXTINCT.
Additional hnRNP-A1-binding sites have been identified within the flanking intron, including the ISS-N1 regions. Collectively, these factors prevent
the identification of the already weak splice sites flanking Exon 7 and promote high levels of exon skipping, ∼90%. A small, but important level
(10%) of SMN2 transcripts include Exon 7 and therefore encodes a fully functional SMN protein.

nucleotide exon.46 Other elements, however, appear
to function only within the context of one gene or
the other.47–52 Remarkably, there are only a handful
of nucleotides that differ between SMN1 and SMN2
and none change the protein coding sequence. One of
these nonpolymorphic nucleotide differences resides
within the SMN Exon 7 (+6) and results in a C–T
transition. While this appears to be a relatively subtle
alteration, it clearly has a dramatic impact upon the
retention of SMN Exon 7. The extreme phenotype
of this nucleotide transition can likely be explained
based upon the disruption of several overlapping
regulatory elements (Figure 5). First, SF2/ASF binds
this region in the SMN1 context and facilitates inclu-
sion of SMN Exon 7.47 In contrast, the SMN2 RNA
is poorly recognized by SF2/ASF and therefore, a pos-
itively acting factor is lost. Second, the C–T transition
creates a novel binding site, heterogeneous nuclear
RNP-A1 (hnRNP-A1).52 Within SMN2 pre-mRNA,
hnRNP-A1 functions as a repressor of Exon 7’s
inclusion, thereby promoting the exon-skipping event.
Finally, an additional regulatory element referred to
as EXTINCT, likely mediated by the secondary struc-
tures within the 5 end of Exon 7, forms in the presence
of the SMN2-specific context.51 Collectively, the loss
of a positively acting factor and the novel presence
of negatively acting factors results in the dramatic
reduction in full-length SMN expression.
Recently, SMN2 has been viewed as an essential
target for therapeutic development because it is
retained in all SMA patients and it has the capacity
to encode a fully functional SMN protein.2,42,53,54
Therefore, strategies that modulate SMN2 Exon 7’s
inclusion and promote full-length expressions have
been at the forefront of SMA translational research.
A variety of modified oligonucleotides, antisense

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 Advanced Review
sequences, and trans-splicing RNAs have been
developed that efficiently increase Exon 7’s inclusion
in SMA patient fibroblasts and more recently in SMA
animal models.49,55–71 As with many central nervous
system disorders, these translational efforts now face
the challenge of moving beyond cell-based assays or
small animals and will need to address critical issues
such as delivery and the therapeutic window.
SMN Protein
SMN is ubiquitously expressed, consistent with its
essential role in survival. The full-length SMN protein
is a 294 amino acid protein with a molecular weight
of 38 kDa and is expressed at high levels in the brain,
spinal cord, kidneys, and liver. Moderate expression
is found in cardiac and skeletal muscle. When the
spinal cord from an SMA type I patient was exam-
ined for SMN protein there was a 100-fold reduction
compared to age-matched standards.72
SMN Biochemical Properties
Through a series of biochemical fractionation experi-
ments, a tightly associated complex has been identified
that is present in all cells and tissues examined to
date: the SMN/Gemin complex12,13,73–78 (Table 1).
This multiprotein complex is nucleated by the SMN
protein and contains a discrete set of interacting pro-
teins called Gemin2–Gemin8, and an additional factor
unrip.74,79–85 A detailed interaction map of these fac-
tors has been developed and while not all factors make
direct contact with the SMN, the complex is remark-
ably stable.86 Interestingly, while the ability of SMN
to self-associate is linked to disease severity and it
has been presumed that SMN exists in a multimerized
state, the precise stoichiometry of SMN and Gemins
is yet to be determined.38,78,87 This macromolecular
complex exhibits localization patterns similar to the
SMN protein: enriched in nuclear gems and diffusely
abundant throughout the cytoplasm.5–10 The primary
exception to this rule would be unrip which appears to
be restricted to the cytoplasm.85 Additionally, in cells
of neuronal origin including cultured neurons or pri-
mary motor neuron cultures, additional SMN/RNP
complexes are present that are independent of Sm
proteins.88–93 Non-snRNP complexes are discussed in
a subsequent section.
Functional and biochemical domains of SMN
have been identified based upon cellular assays as well
as insight derived from intragenic SMN point muta-
tions from SMA patients4,125,126 (Figure 1). The SMN
protein has an amino-terminal nucleic acid-binding
domain in the Exon 2a 2b region which overlaps
with the Gemin2 interaction regions.127–129 Exon 3
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contains a Tudor domain which mimics a truncated
Smith protein fold with five β-pleated sheets and
appears to nucleate the formation of newly formed
Smith core proteins.130–134 Exon 5 contains a polypro-
line motif similar to the VASP protein family and is
responsible for mediating interactions with the pro-
filin protein.98,130–136 Profilin binds ATP-actin at a 1:1
ratio to sequester it in an inactive globular form. SMN
protein contains an evolutionarily conserved Y/G box
which mediates self-association through Exon 6.87,137
Exon 2 also contributes to self-association through a
separate oligomerization domain.129,138 Exon 7 con-
fers stability and contains a proposed cytoplasmic
localization motif ‘QNQKE’.38,93,139–143 This motif is
sufficient to dramatically alter a heterologous nuclear
protein and it is clear that this sequence can target
proteins to the cytoplasm. Refined characterization
of the stability and localization conferred by the
SMN tail region determined that a minimal (4–6
amino acids), nonspecific sequence was sufficient to
at least partially compensate for much of the native
sequence.139–142 The C-terminally truncated SMN7
mRNA stops translation at the next available termi-
nation codon within Exon 8. The similarities between
the two isoforms are an equivalent mRNA half-life
and the nuclear export of the SMN7 and full-
length mRNAs.144 The failure of the SMN7 protein
to subsume those functions attributed to full-length
SMN protein devolves primarily from its inability to
oligomerize, an essential precondition for all SMN-
associated functions. The inability to be incorporated
into the appropriate RNP complexes presumably ren-
ders the protein less stable.
SMN in snRNP Assembly
Eukaryotic messenger pre-mRNA splicing is an essen-
tial step in nearly all gene expressions, impacting the
diversity of the transcriptome, and allowing for tem-
poral, spatial, and developmental control of various
gene products. The general splicing machinery largely
consists of small nuclear RNA (snRNA)/protein com-
plexes comprised of snRNAs, Smith core proteins, and
additional conserved protein factors. These RNPs will
perform the catalytic reactions necessary to identify
the intervening intron sequences and the ligation of
the exon sequences. While SMN was initially believed
to have a direct role in pre-mRNA splicing,123,145 it
is now clear that SMN is intimately involved in the
formation and maturation of snRNP particles and
therefore performs an essential, yet indirect, role in
pre-mRNA splicing.4,12,13,76
The formation of a snRNP particle starts with
the polymerase II-mediated transcription of one of the
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 WIREs RNA SMN in spinal muscular atrophy and snRNP biogenesis

TABLE 1 Previously Identified Survival Motor Neuron (SMN) Interacting Proteins Are Shown
SMN Interacting Factor Function Direct/Indirect Motifs References
Core SMN/Gemin components
SMN snRNP assembly Direct Tudor, Y/G box 74
Gemin2/SIP1 snRNP assembly Direct – 74
Gemin3/DDX20/DP103 snRNP assembly Direct DEXDc, ELICc 81
Gemin4/GIP1 snRNP assembly Indirect Leucine zipper 82
Gemin5/p175 snRNP assembly Direct WD40 83
Gemin6 snRNP assembly Indirect – 84
Gemin7/SIP3 snRNP assembly Indirect RG-rich 79
Gemin8 snRNP assembly Direct – 80
Unrip snRNP assembly Indirect WD40 85
snRNP assembly/maturation
Sm proteins/Sm B/B ,D1–3,E,F,G snRNP assembly Direct/indirect Sm-fold, RG-rich 74,94
Sm-like proteins/Lsm 10,11 snRNP assembly Direct/indirect Sm-fold, RG-rich 94–96
Snurportin and importin β snRNP nuclear import Direct/indirect HEAT repeat 96
TGSI (trimethylguanosine synthase1) Hypermethylation of m7 G cap Direct – 97
Actin dynamics
Profilin Actin transport Direct – 98
Plastin 3/ T-plastin Actin bundling Indirect EFh, CH 99
RNPs/RNA metabolism
hnRNP-Q RNA metabolism Direct RRM, RGG 88,100
hnRNP-R RNA metabolism Direct RRM, RGG 88,100
hnRNP-U RNA metabolism Direct SAP, SPRY, RGG 6
FMRP RNA metabolism Direct KH 101
KSRP/FBP2/ZBP2/MARTA1 RNA metabolism Direct KH, RGG 102
U1A Pre-mRNA splicing ND RRM 74
Galectin 1 and 3/LGALS1 Pre-mRNA splicing Indirect GLECT 103
RNA helicase A Transcription Direct DSRM, DEXD, HELIC, RGG 104
RNA polymerase II Transcription Direct – 104
Rpp20 tRNA/rRNA metabolism Direct – 105
Nucleolin rRNA metabolism Indirect RRM, RGG box 106
ISG20 ssRNA degradation ND EXOIII 107
TIAR Stress granules RRM 108,109
TDP43 Pre-mRNA splicing/transcription ND RRM 109
NFAR1/2 RNA metabolism Direct DZF, DSRM 110
Additional factors
p80 Coilin Cajal body recruitment Direct RGG 111
mSin3A Transcription ND PAH 112
EWS Transcription Direct RGG 113
Papilloma virus E2 Transcription Direct DNA binding 114
EBNA2 Transcription/viral replication Direct Acidic activation domain 115
MVM NS1/NS2 Transcription/viral replication Direct Nucleic acid binding 116,117
p53 Apoptosis Direct DNA binding 118
Osteoclast-stimulating factor Signal transduction Direct SH3, ANK 119

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TABLE 1 Continued
SMN Interacting Factor Function Direct/Indirect Motifs References
USP9X (ubiquitin-specific protease 9) Deubiquitylating enzyme ND – 120
PPP4 (protein phosphatase 4) Ser/Thr protein phosphatase Indirect PP2Ac 121
FGF2 (fibroblast growth factor 2) Growth factor Direct FGF 122
HSC70 Protein chaperone ND – 75
ZPR1 Splicing/transcription Direct Zinc finger domain 123
BAT3 Apoptosis ND UBQ domain 124
UNC119 Photoreceptor synaptic protein ND GMP-PDE 124
RIF1 Transcriptional repressor ND – 124
GDF9 Growth factor ND TGFB 124
COPS6 Ubiquitin cascade ND JAB/MPN 124

The function of the cellular protein is included when known. However, the functional connections to the interacting proteins have not been determined in all
cases.

snRNAs (Figure 6). For the majority of pre-mRNA
splicing events, the snRNAs of interest are the
uridine-rich U1, U2, U4, U5, and U6.146 Within the
nucleus, a 7-methylguanosine (m7 G) cap is added to
the nascent transcripts,147,148 which in turn, leads
to the formation of a cap complex consisting of
snRNA, PHAX, XPO1/CRM1, and cap-binding com-
plex (CBC).149–156 Cap formation and the processing
of the 3 end of the RNA are the molecular triggers
for nuclear export.11 Collectively, these factors facil-
itate the nuclear export of the m7 G-capped snRNA
into the cytoplasm through a heterodimeric CBC con-
sisting of cap-binding protein 20 and 80 (CBP20
and CBP80).151,152,155,157 Once in the cytoplasm,
the export complex dissociates in a GTP-dependent
step.155
Within the cytoplasm, a series of proteins
referred to as Smith core antigens or Sm protein
forms an ordered protein structure resembling a hep-
tameric ring.158 The Sm proteins common to all
snRNPs are encoded by seven genes: Sm B/B , D1,
D2, D3, E, F, and G.146 B and B are derived
from the same gene but are generated through an
alternative splicing mechanism.159 While Sm proteins
can thermodynamically self-assemble in vitro, the
unassembled Sm proteins within the cytoplasm cannot
merely assemble upon the recently imported snRNA.
To initiate the heptameric formation of the Sm pro-
teins, the first step involves the interaction of the Sm
proteins with the chloride conductance regulatory pro-
tein (pICln).12,94,95,160–162 As these are RNA-binding
proteins, this step is at least partly designed to pre-
vent spurious RNA binding by the Sm proteins. At
this stage, the Sm proteins retain a relatively low
affinity for snRNAs. pICln, however, is a compo-
nent of a macromolecular complex referred to as
the protein arginine methyltransferase 5 (PRMT5) or
methylosome.12,94,95,160–162 The interaction between
the Sm proteins and PRMT5 and an additional factor,
PRMT7, results in the symmetrical dimethylation of
arginine residues in SmB, SmD1, and SmD3, thereby
increasing the binding affinity between SMN and the
Sm proteins.163,164 Prior to binding to the snRNA, the
Sm proteins exist in four identifiable complexes: (1) a
hexameric complex consisting of Sm E, F, G dimers;
(2) Sm D3 and B; (3) a Sm B complex; and (4) a Sm
D1, D2, and hexameric complex consisting of Sm E,
F, and G dimers.158
At this stage, the SMN/Gemin complex interacts
with the Sm proteins and mediates the formation of
a ring-like structure around the highly conserved Sm
site on the snRNA.13,75,76,164–166 The region of SMN
encoded by Exon 3 includes a Tudor domain which
serves as a binding site for the Sm proteins.130–134
Remarkably, while the SMN Tudor region as well as
Gemins6 and 7 do not contain any primary amino
acid sequence similarities to the Sm proteins, each
of these proteins are capable of forming structural
motifs that closely resemble the Sm or Sm-like
protein folds.133,167–169 The heterodimeric structure of
Gemin6 and Gemin7 forms a five-stranded antiparallel
β sheet flanked by an amino-terminal helix, and
while not conclusively demonstrated, may facilitate
the loading of individual Sm proteins into the complex
and the eventual closure of the ring formation.169,170
The Sm site within the snRNA serves as a nucle-
ation site for the binding and formation of the hep-
tameric Sm ring.167 In this ATP-dependent reaction,
there are interactions between the Sm proteins, SMN,
and the Gemin proteins.13,75,76,164–166 The Gemin5
protein appears to provide specific guidance to the Sm
proteins as it is responsible for specifically identifying
target RNAs that contain the Sm-binding site.171,172

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 WIREs RNA SMN in spinal muscular atrophy and snRNP biogenesis

Sm
proteins Cajal body
Gem
RNAPII
5′ 3′
PRMT7
UsnRNA
PRMT5 m3G Sm

Sm
methylation SMN/Gemin
complex
Sm

SMN/Gemin
complex

Snurportin

Sm
m7G Sm
Importin-β
7G Sm
m

FIGURE 6 | Overview of the survival motor neuron (SMN) functions in the assembly of mature spliceosome particles. Small nuclear RNAs
(snRNAs) are transcribed via polymerase II-dependent expression. The presence of the m7 G cap initiates binding by the nuclear export complex
consisting of PHAX, XPO1/CRM1, RAN, and both subunits of the cap-binding complex (CBC). Upon entering the cytoplasm, the nuclear export
complex dissociates. Within the cytoplasm, several complexes consisting of partially assembled, snRNA-free Sm proteins reside in the cytoplasm:
SmD1/SmD2; SmB/B /SmD3; and SmE/SmF/SmG. Prior to binding the SMN complex, PRMT5 and PRMT7 specifically methylate the C-terminal tails of
SmB, SmD1, and SmD3, thereby enhancing the affinity for the SMN complex. The SMN/Gemin/unrip complex binds the methylated Sm complex,
allowing the specific recruitment of snRNAs. The Sm complex is guided onto the Sm-binding site on the snRNA by Gemin5 at which point the
heptameric ring structure is formed. The SMN/Gemin/small nuclear ribonucleoprotein (snRNP) complex interacts with trimethylguanosine synthetase
1 (TGS1) which results in the hypermythylation of the m7 G cap and the formation of the m3 G cap. The nuclear import complex consisting of
snurportin and importin-β interacts with the m3 G cap and the SMN complex and transports the entire complex into the nucleus. Entry into the
nucleus results in the release of the import complex, the SMN complex, and the snRNP’s initial transitions through the Cajal body.

Once the Sm core has assembled, additional
maturation steps are required prior to nuclear import.
The snRNA 5 m7 G cap undergoes a series of tran-
sitions, first of which involves the hypermethylation
by trimethylguanosine synthetase 1 (TGS1), result-
ing in the formation of a 2,2,7-trimethyl-guanosine
(TMG or m3 G) cap.173 This complex is now primed
for nuclear import. The TMG cap and the assembled
Sm core serve as two separate nuclear localization
signals each utilizing the importin β nuclear import
receptor.174 While nuclear localization signals are
present within several members of the SMN/Gemin
complex, the direct involvement of these elements
has not been fully characterized. The SMN/Gemin/Sm
core complex then interacts with snurportin-1 and the
complex transitions the nuclear membrane.96
Within the nucleus, the SMN/Gemin/Sm core
complex is bound by RanGTP, triggering the release
of the U-snRNP.96,175 Snurportin-1 is quickly shuttled
back into the cytoplasm as it becomes bound by
XPO1/CRM1. The U-snRNP is now able to transition
between the nucleoplasm and several discrete
subnuclear structures, the first of which are Cajal
bodies.176–178 While the precise role of the Cajal body
in the function or redirection of the U-snRNP remains
unclear, these regions accumulate the newly imported
U-snRNPs. Importantly, however, these are not sites
of active transcription or RNA processing. Rather,

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 Advanced Review
U-snRNPs are subsequently transported to additional
subdomains that are the sites intimately associated
with pre-mRNA splicing, including perichromatin
fibrils and interchromatin granule clusters.11,178–182
Connecting SMA to snRNP-Associated
Defects: Major Versus Minor Pathways
One of the vexing questions in SMA focuses upon
the SMN-associated function in disease development.
Two primary, although not necessarily, mutually
exclusive views of the SMN role(s) which, when
lost, contribute most to SMA pathology have recently
emerged4 : snRNP biogenesis and axonal mRNA
trafficking. The snRNP assembly activity of the
SMN/Gemin complex is clearly involved in a general
cellular function required for survival of any cell or
tissue type. So how could this general function account
for the motor neuron-specific pathology observed in
most SMA cases? Are specific transcripts altered from
motor neuron-specific genes? Recent work has begun
to shed light on one potential molecular mechanism
that may explain the tissue specificity: the minor
spliceosome.183–185 The overwhelming majority of
introns are excised using a relatively abundant form of
the spliceosomal snRNP population referred to as the
‘major’ pathway or the U2-dependent spliceosome.183
In this RNP complex, U1, U2, U4/U6, and U5 snRNPs
mediate the identification and excision of intron
sequences. In contrast, an alternative spliceosomal
complex exists that is responsible for the excision
of introns in ∼600–700 genes, or less than 1% of
introns within the transcriptome.183–185 This pathway,
referred to as the ‘minor’ pathway or U12-dependent
pathway, consists of U11, U12, U4atac/U6atac, and
U5 snRNPs. U5 snRNP is unique in that it is common
among both pathways. Unlike U2-dependent introns
which typically contain the canonical GT/AG splice
sites, the U12-dependent introns contain a 5 AT splice
site and an AC 3 splice site.183,186 Additionally, for
many of the AT/AC introns, the typical branch site
is less conserved than the comparable motif in U2-
dependent introns and it is believed that the 3 splice
site and the branch site may function as a single entity
rather than two separate regulatory domains. Con-
sistent with this, it appears that greater regulatory
properties are associated with the 5 splice site in U12
introns.187–189
In SMA animal models, SMN levels and snRNP
assembly/activity during development are lower than
that seen in control animals.3,190 Analysis of animals
with different forms of disease revealed an inverse
correlation between disease severity and snRNP
activity.190 This work suggested that as SMN levels
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would ensue, a vast array of genes could be expected to
be impacted. Recent work has demonstrated patho-
logically low levels of SMN results in general gene
expression defects.191 In one study that examined a
variety of tissues near end-stage in a severe model of
SMA, expression anomalies were detected in nearly
all tissues and across broad gene families. One poten-
tial caveat is that these animals typically exhibit a
life span of 13–15 days and tissues were examined at
11 days; therefore, some of these differences may be
attributable to the debilitated state of the animals.191
Additionally, recent work by Baumer et al. revealed
that widespread modifications in gene expression and
splicing are associated with late-symptomatic stages
of SMA and are not detectable earlier in development,
suggesting that global gene expression alterations
could be due to secondary effects rather than a SMN-
specific deficiency.192 While these results are difficult
to reconcile with motor neuron-specific defects, it is
worth noting that in very severe forms of SMA, both
human and murine, (Smn−/− ; SMN2+/+ and Smn−/− ;
SMN2+/+ ; SMN7 mice), additional developmen-
tal defects have been detected within the brain and
cardiac tissue.193–196 Therefore, in extremely low lev-
els of SMN, it may be that a threshold is breached
that goes beyond that necessary to cause diseases in
a motor neuron-restricted manner, and results in a
more far-reaching disease.
Similarly, snRNP assembly levels were shown
to be broadly reduced in several animal models with
additional subtle differences detected when specific
snRNP profiles were examined.190 In spinal cord and
brain extracts from severe SMA pups, many of the
snRNPs were reduced, however, there was a further
reduction in the U11 and U12 snRNPs.190 This sug-
gested that while there was a global reduction in
snRNP activity, there was an even greater preferential
reduction in the minor snRNP population.
Interestingly, U12 introns are not randomly
scattered and evenly distributed throughout the
genome.189,197 In fact, an early analysis of the AT/AC
introns identified a cluster of voltage-gated ion chan-
nel genes that were enriched for U12 introns.189,197
As, these genes are associated with axonal function
and mutation, in many of these genes give rise to neu-
romuscular and neurodegenerative disorders, splicing
defects in these genes could provide a mechanistic
model explaining how splicing defects might result
in a neuronal pathology. Not only did these genes
contain a U12 intron, there were many instances in
which multiple minor introns were contained within
a single gene.197 A scenario could be envisioned in
which SMN is required for general snRNP assem-
bly, however, as SMN levels decrease, the pool of
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 WIREs RNA SMN in spinal muscular atrophy and snRNP biogenesis

existing minor spliceosome snRNPs would be the first
impacted because the concentration of these factors is
lower than the major spliceosome snRNPs. Genes with
a single minor intron could be adversely impacted,
but genes harboring multiple minor introns—such as
the voltage-gated ion channel genes—could be pref-
erentially misregulated. To date, splicing defects in
specific U12-dependent genes in SMA contexts have
not been identified; however, this line of investigation
would provide important insight into the molecular
basis of SMA as well as provide additional targets for
therapeutic development.
SMN and Axonal RNP Complexes
While the primary focus of this review is on the role
of SMN in snRNP maturation, it is also instructive to
examine the SMNs role in the formation of additional
RNPs as it has been described in a variety of other
functional RNP complexes. Another RNP-associated
role for SMN has proposed that may account for dis-
ease development: axonal mRNA transport.198 SMN
has been identified with a complex found within
axons that contains non Sm-related proteins, including
hnRNPs Q and R (hnRNP-Q/R) as well as Z-DNA-
binding protein 1/2 (ZBP1, ZBP2)88–90,92,93,136,199,200
(Figure 7). These factors have been shown to facil-
itate the active transport of a subset of mRNAs,
including β-actin, KSRP/ZBP2/MARTA1,102 FBP,201
hnRNP-R, and hnRNP-Q88,100,200 to the axon growth
cone allowing for localized translation.93,200,202–206
This is accomplished through a direct association with
the 3 UTR of β-actin mRNA known as the zip code
sequence. In cultured motor neurons derived from a
SMA mouse model, β-actin mRNA and protein was
significantly reduced in axonal growth cones.88,200,207
This molecular defect correlated with smaller growth
cone size and shorter axons. Additionally, the knock-
down of SMN in a zebrafish model resulted in specific
motor neuron pathway finding defects and presynaptic
neuromuscular junction (NMJ) defects.208,209 How-
ever, studies in several mouse models have shown
that the majority of NMJs appear to develop nor-
mally, albeit slower, suggesting that defects in axon
outgrowth and related synaptogenesis pathways are
unlikely to be the primary development defect in
SMA in vivo.210,211 Interestingly, the reintroduction
of SMN-free snRNP particles was shown to over-
come the SMN-associated defects in zebrafish motor
neurons.212
Further evidence for SMN in an axonal-specific
function has been reported by the identification of

Well developed
hnRNP-Q/R NMJ
ZPB
Cell body

SMN
β-actin mRNA

Unaffected motor neuron

Poorly developed
hnRNP-Q/R NMJ
ZPB
Cell body

β-actin mRNA

SMA motor neuron

FIGURE 7 | Proposed role for the survival motor neuron (SMN) in neurons involves a novel ribonucleoprotein (RNP) complex. Within axons, a pool
of SMN exists that is not complexed with Sm proteins and the entire Gemin complex. Additional SMN–RNP forms that contain hnRNP-Q and -R (blue)
are ZPB (green) and β-actin mRNA. The active transport of this complex results in the accumulation of β-actin mRNA and protein at the distal end of
the axon and at the neuromuscular junction (NMJ). In the spinal muscular atrophy (SMA; bottom panel), low levels of the SMN result in reduced
levels of β-actin accumulation and poorly developed NMJs.

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 Advanced Review
a SMA modifying gene called Plastin 3/T-plastin
(PLS3).99 PLS3 is involved in actin dynamics and
aids in the stabilization of filamentous actin, thereby
increasing the pool of F-actin.213,214 In discordant sib-
lings, increased expression of PLS3 correlated with
decreased disease severity in females.99 Defects in
cultured motor neurons from SMA mice or from
SMN-depleted zebrafish could also be overcome by
the transient expression of PLS3.99 While additional
work such as SMA transgenic PLS3 mice will need to
be developed, this work supports the notion that actin
dynamics plays a key role in SMA development. Addi-
tionally, administration of a synthetic peptide that
inhibits Rho-kinase (ROCK) significantly corrected
the NMJ defects and extended the survival of a milder
model of SMA.215,216 Through this pathway, RhoA
has been shown to perform important roles in the
regulation of actin dynamics. Collectively, this work
illustrates the potential importance of actin transport
and actin dynamics in the development of SMA.
CONCLUSION
SMN is clearly a multifunctional protein that serves as
a nucleation point for a variety of RNPs. A number of
intragenic mutations have been identified within SMA
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