MOLECULAR AND CELLULAR BIOLOGY, July 2005, p. 5543–5551
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 25, No. 13
The Survival of Motor Neurons Protein Determines the Capacity for
snRNP Assembly: Biochemical Deficiency
in Spinal Muscular Atrophy
Lili Wan, Daniel J. Battle, Jeongsik Yong, Amelie K. Gubitz, Stephen J. Kolb, Jin Wang,
and Gideon Dreyfuss*
Howard Hughes Medical Institute, Department of Biochemistry & Biophysics, University of Pennsylvania School
of Medicine, Philadelphia, Pennsylvania 19104-6148
Received 14 February 2005/Returned for modification 23 March 2005/Accepted 30 March 2005
Reduction of the survival of motor neurons (SMN) protein levels causes the motor neuron degenerative
disease spinal muscular atrophy, the severity of which correlates with the extent of reduction in SMN. SMN,
together with Gemins 2 to 7, forms a complex that functions in the assembly of small nuclear ribonucleoprotein
particles (snRNPs). Complete depletion of the SMN complex from cell extracts abolishes snRNP assembly, the
formation of heptameric Sm cores on snRNAs. However, what effect, if any, reduction of SMN protein levels,
as occurs in spinal muscular atrophy patients, has on the capacity of cells to produce snRNPs is not known.
To address this, we developed a sensitive and quantitative assay for snRNP assembly, the formation of
high-salt- and heparin-resistant stable Sm cores, that is strictly dependent on the SMN complex. We show that
the extent of Sm core assembly is directly proportional to the amount of SMN protein in cell extracts.
Consistent with this, pulse-labeling experiments demonstrate a significant reduction in the rate of snRNP
biogenesis in low-SMN cells. Furthermore, extracts of cells from spinal muscular atrophy patients have a lower
capacity for snRNP assembly that corresponds directly to the reduced amount of SMN. Thus, SMN determines
the capacity for snRNP biogenesis, and our findings provide evidence for a measurable deficiency in a
biochemical activity in cells from patients with spinal muscular atrophy.
The process of pre-mRNA splicing is carried out by a mac-
romolecular complex, the spliceosome, the major components
of which are the U1, U2, U5, and U4/U6 small nuclear ribo-
nucleoprotein particles (snRNPs) (18, 34, 47). Each of the
snRNPs (except for U6) is composed of one snRNA molecule,
a set of seven common proteins, and several proteins that are
specific to individual snRNAs (18, 27, 28, 47). SnRNP biogen-
esis begins with the transcription of the snRNAs in the nucleus
followed by their nuclear export to the cytoplasm, where the
major assembly process of the snRNPs takes place. The com-
mon proteins, called Sm proteins, B/B?, D1, D2, D3, E, F, and
G, are arranged into a stable heptameric ring, the Sm core, on
a uridine-rich sequence motif, the Sm site, of the snRNAs (1,
2, 19, 41). The assembly of Sm cores is required for the sub-
sequent modification of the 7-methyl guanosine cap of
snRNAs into a 2,2,7-trimethyl guanosine cap as well as for the
stability and function of the snRNPs (30, 38). Properly assem-
bled and modified snRNPs are then imported into the nucleus,
where additional snRNP-specific proteins associate to form
fully functional snRNPs (10, 11, 13, 30, 31, 47).
Earlier studies have shown that snRNP assembly readily
occurs in vitro with purified total snRNP proteins (TPs) and
snRNAs in an ATP-independent manner and without require-
ment for non-snRNP proteins (39, 40, 43). However, reconsti-
tution of snRNPs in extracts from Xenopus laevis eggs and
mammalian cells requires ATP (21, 32, 33, 37, 44), suggesting
that snRNP assembly might be regulated by additional factors
in vivo. Studies on a macromolecular complex containing the
survival of motor neurons (SMN) protein indicated that the
SMN complex is required for the ATP-dependent snRNP as-
sembly (3, 9, 32, 33, 36, 37, 49). SMN is the protein product of
the gene responsible for spinal muscular atrophy (SMA), a
common and often fatal genetic disorder in which motor neu-
rons in the spinal cord degenerate (6, 8, 15, 22). Based on the
age of onset and the severity of the disease, SMA is clinically
classified into three types: the severe type I, the moderate type
II, and the mild type III. Studies on SMA patient-derived cell
lines have shown that the severity of SMA clinical phenotypes
is closely linked to the degree of reduction of SMN protein
levels (7, 23).
Immunodepletion or antibody inhibition of the SMN com-
plex in vitro demonstrated that the SMN complex is required
for snRNP assembly (32, 33, 37). However, how much the
SMN protein as well as individual Gemins contribute to
snRNP assembly and what happens in SMA patients’ cells,
where the amount of SMN protein is reduced to various de-
grees, have not been determined. Current methods using gel
mobility shift assay to monitor snRNP assembly are not suit-
able for quantitative analysis, due to the heterodisperse migra-
tion of large RNP complexes on native gels. To assess the
relationship between the amount of SMN and the activity of
Sm core assembly in cells and to facilitate further studies on
the mechanism of SMN complex function, we developed a
sensitive and quantitative assay for snRNP assembly.
The assay is based on the isolation of high-salt- and heparin-
resistant Sm cores formed on biotin-labeled snRNAs with
* Corresponding author. Mailing address: Howard Hughes Medical
Institute, Department of Biochemistry & Biophysics, University of
Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-
6148. Phone: (215) 898-0398. Fax: (215) 573-2000. E-mail: gdreyfuss
magnetic beads bearing anti-Sm antibodies. The amount of
snRNPs assembled in the reaction is then determined by lu-
minescence detection of the biotin molecules on the snRNAs.
Importantly, we show that the extent of assembly is directly
dependent on the amount of the SMN complex in cell extracts
and that extracts from cells of SMA patients have a lower
capacity for snRNP assembly, proportional to the reduction of
SMN protein in these cells. In vivo pulse-labeling experiments
demonstrate that the rate of biogenesis of the major snRNPs is
strongly reduced in cells with low SMN. These findings provide
evidence for a deficiency in a specific biochemical activity in
SMA patients and present a powerful method for studying the
activity of the SMN complex.
MATERIALS AND METHODS
In vitro transcription of RNAs. Plasmids for in vitro transcription of snRNAs
were described elsewhere (10, 13, 16, 30). For radiolabeling of RNAs, in vitro
transcription was carried out in the presence of [32P]UTP as previously described
(48). Biotin-labeled RNAs were produced according to the suggestions of the
manufacturer (Ambion) with the modification that 5 mM biotin-UTP (Roche)
and 2.5 mM UTP were present. All of the labeled RNAs were purified by
electrophoresis on 7 M urea–6% polyacrylamide gels, precipitated with ethanol,
and resuspended in nuclease-free water. The concentrations of the biotin-labeled
RNAs were determined by absorbance at 260 nm.
Cell lines and cell culture maintenance. The maintenance of the S5 cell line,
which is a chicken DT40 cell line with targeted disruption of the SMN gene, has
been described previously (45). Epstein-Barr virus-transformed lymphoblast cell
lines derived from a 6-month-old SMA type I patient (GM10684) and an age-
and gender-matched individual with a syndrome unrelated to SMA as a control
(GM12497) were obtained from Coriell Cell Repositories and maintained in
RPMI 1640 medium (Gibco BRL) containing 10% fetal bovine serum (HyClone)
and 1% penicillin-streptomycin (Gibco BRL). Primary fibroblast cell lines from
four SMA type I patients (GM00232, GM09677, GM03813, and GM03815), one
heterozygous carrier (GM03814), and two apparently healthy controls
(GM08333 and GM00498) were also obtained from Coriell Cell Repositories.
They were maintained in minimal essential medium (Gibco BRL) containing
15% fetal bovine serum, 2 mM L-glutamine, and 1% penicillin-streptomycin.
Preparation of cytoplasmic extracts from cultured cells. For each sample in
the assays shown, the same number (?4 ? 107) of cells were harvested and
washed twice with phosphate-buffered saline. For the SMA fibroblast cell lines,
cells were used at an early and identical passage stage. Cytoplasmic extracts
competent for snRNP assembly were prepared as described previously (37). The
protein concentrations of the various extracts were determined using the Bio-
Rad protein assay (Bio-Rad). All of the extracts were adjusted to the same final
protein concentration (?15 mg/ml for extracts prepared from HeLa cells, S5
cells, or SMA lymphoblastoid cells, and 3 mg/ml for extracts of SMA fibroblast
cells) and were quickly frozen in liquid nitrogen and stored in aliquots at ?80°C.
Assay for in vitro assembly of snRNPs. For in vitro Sm core assembly on
32P-labeled snRNAs in cytoplasmic extracts, the reactions were carried out at
30°C for 1 h using standard assembly reaction conditions (37). Subsequently, half
of the reaction mixtures was loaded onto native gels for electrophoretic mobility
shift assays as described previously (37). The other half of the samples was
immunoprecipitated with the anti-Sm monoclonal antibody Y12 (24), and the
immunoprecipitated RNAs were isolated and analyzed by electrophoresis on 7
M urea–8% polyacrylamide gels.
For quantitative in vitro assembly assays on magnetic beads, cytoplasmic ex-
tracts were prepared and used for assembly on biotin-labeled snRNAs using the
standard reconstitution conditions in 96-well plates. Following the reactions, Y12
antibodies immobilized onto the magnetizable Dynabeads protein A (Dynal
Biotech ASA, Oslo, Norway) in 100 ?l of RSB-500 buffer (10 mM Tris-HCl, pH
7.5, 500 mM NaCl, 2.5 mM MgCl2) containing 2 mg/ml heparin, 0.1% NP-40, and
0.2 U/?l RNasin RNase inhibitor (Promega) were added to each well. Immu-
noprecipitations in the 96-well plates were carried out with gentle mixing at 750
rpm in a Thermomixer (Eppendorf, Germany) at 30°C for 1 h. The plates were
subsequently transferred to a Kingfisher 96 magnetic particle processor (Thermo
Labsystems, Vantaa, Finland) for automatic washing of the Dynabeads in each
well with 200 ?l of wash buffer (RSB-500, 0.1% NP-40) five times. After the last
wash, the beads that were bound with Y12-immunoprecipitated snRNPs were
then resuspended in 120 ?l of wash buffer containing 0.08 ?g/ml horseradish
peroxidase-conjugated NeutrAvidin (Pierce). Following incubation at 30°C for
1 h with gentle mixing, the beads in each well were again washed five times by the
Kingfisher 96 magnetic particle processor and finally resuspended in 150 ?l of
SuperSignal enzyme-linked immunosorbent assay (ELISA) Femto substrate
working solution (Pierce). The plates were transferred to a Wallac Victor2
multilabel plate reader (Perkin-Elmer) for luminescence measurements at 495
nm. The resulting data were analyzed with Microsoft Excel.
FIG. 1. Analysis of in vitro-assembled snRNPs by the mobility gel
shift assay and by the magnetic beads assay. (A) [32P]UTP-labeled U1,
U1?Sm, U1A3, U4, U4?Sm, U5, or U5?Sm snRNA was mixed with
HeLa cytoplasmic extracts (CE) containing 25 ?g total proteins (?
lanes) or with buffer only (? lanes) for in vitro assembly of Sm cores.
The reaction mixtures were analyzed by electrophoresis on 6% native
polyacrylamide gels. The brackets on the right indicate the positions of
assembled Sm cores and the free RNAs. The complex that results from
the binding of the U1-specific protein U1A to stem-loop 2 of the U1
snRNA is marked on the left. The band indicated by an asterisk is
likely to be U1A/Sm core complexes. (B) The same reaction mixtures
as used in panel A were immunoprecipitated by Y12 bound to protein
A-Sepharose beads. RNAs were isolated from the bound fractions and
analyzed by electrophoresis on 7 M urea–8% polyacrylamide gels. The
RNAs that migrate at different positions on the gel are indicated on
the left. (C) SnRNAs were produced by in vitro transcription in the
presence of biotin-UTP instead of [32P]UTP, and similar assembly
reactions as in A were carried out with these biotinylated RNAs or
without any RNA using either HeLa cytoplasmic extracts (? lanes) or
buffer (? lanes). The amount of the Sm cores assembled on these
RNAs was assessed by the magnetic beads assay as depicted in Fig. 2.
The error bars, which for some data points are too small to be seen on
the figure, represent standard deviations from three independent ex-
5544 WAN ET AL.MOL. CELL. BIOL.
atrophy disease gene product, SMN, and its associated protein SIP1 are in a
complex with spliceosomal snRNP proteins. Cell 90:1013–1021.
27. Luhrmann, R. 1990. Functions of U-snRNPs. Mol. Biol. Rep. 14:183–192.
28. Luhrmann, R., B. Kastner, and M. Bach. 1990. Structure of spliceosomal
snRNPs and their role in pre-mRNA splicing. Biochim. Biophys. Acta 1087:
29. Mangin, M., M. Ares, Jr., and A. M. Weiner. 1985. U1 small nuclear RNA
genes are subject to dosage compensation in mouse cells. Science 229:272–
30. Mattaj, I. W. 1986. Cap trimethylation of U snRNA is cytoplasmic and
dependent on U snRNP protein binding. Cell 46:905–911.
31. Mattaj, I. W., W. Boelens, E. Izaurralde, A. Jarmolowski, and C. Kambach.
1993. Nucleocytoplasmic transport and snRNP assembly. Mol. Biol. Rep.
32. Meister, G., D. Buhler, R. Pillai, F. Lottspeich, and U. Fischer. 2001. A
multiprotein complex mediates the ATP-dependent assembly of spliceoso-
mal U snRNPs. Nat. Cell Biol. 3:945–949.
33. Meister, G., and U. Fischer. 2002. Assisted RNP assembly: SMN and
PRMT5 complexes cooperate in the formation of spliceosomal UsnRNPs.
EMBO J. 21:5853–5863.
34. Nilsen, T. W. 2003. The spliceosome: the most complex macromolecular
machine in the cell? BioEssays 25:1147–1149.
35. Pellizzoni, L., B. Charroux, J. Rappsilber, M. Mann, and G. Dreyfuss. 2001.
A functional interaction between the survival motor neuron complex and
RNA polymerase II. J. Cell Biol. 152:75–85.
36. Pellizzoni, L., N. Kataoka, B. Charroux, and G. Dreyfuss. 1998. A novel
function for SMN, the spinal muscular atrophy disease gene product, in
pre-mRNA splicing. Cell 95:615–624.
37. Pellizzoni, L., J. Yong, and G. Dreyfuss. 2002. Essential role for the SMN
complex in the specificity of snRNP assembly. Science 298:1775–1779.
38. Plessel, G., U. Fischer, and R. Luhrmann. 1994. m3G cap hypermethylation
of U1 small nuclear ribonucleoprotein (snRNP) in vitro: evidence that the
U1 small nuclear RNA-(guanosine-N2)-methyltransferase is a non-snRNP
cytoplasmic protein that requires a binding site on the Sm core domain. Mol.
Cell. Biol. 14:4160–4172.
39. Raker, V. A., K. Hartmuth, B. Kastner, and R. Luhrmann. 1999. Spliceoso-
mal U snRNP core assembly: Sm proteins assemble onto an Sm site RNA
nonanucleotide in a specific and thermodynamically stable manner. Mol.
Cell. Biol. 19:6554–6565.
40. Raker, V. A., G. Plessel, and R. Luhrmann. 1996. The snRNP core assembly
pathway: identification of stable core protein heteromeric complexes and an
snRNP subcore particle in vitro. EMBO J. 15:2256–2269.
41. Stark, H., P. Dube, R. Luhrmann, and B. Kastner. 2001. Arrangement of
RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein
particle. Nature 409:539–542.
42. Sumner, C. J., T. N. Huynh, J. A. Markowitz, J. S. Perhac, B. Hill, D. D.
Coovert, K. Schussler, X. Chen, J. Jarecki, A. H. Burghes, J. P. Taylor, and
K. H. Fischbeck. 2003. Valproic acid increases SMN levels in spinal muscular
atrophy patient cells. Ann. Neurol. 54:647–654.
43. Sumpter, V., A. Kahrs, U. Fischer, U. Kornstadt, and R. Luhrmann. 1992. In
vitro reconstitution of U1 and U2 snRNPs from isolated proteins and
snRNA. Mol. Biol. Rep. 16:229–240.
44. Temsamani, J., M. Rhoadhouse, and T. Pederson. 1991. The U2 small
nuclear ribonucleoprotein particle associates with nuclear factors in a pre-
mRNA independent reaction. J. Biol. Chem. 266:20356–20362.
45. Wang, J., and G. Dreyfuss. 2001. A cell system with targeted disruption of
the SMN gene: functional conservation of the SMN protein and dependence
of Gemin2 on SMN. J. Biol. Chem. 276:9599–9605.
46. Warner, J. R. 1999. The economics of ribosome biosynthesis in yeast. Trends
Biochem. Sci. 24:437–440.
47. Will, C. L., and R. Luhrmann. 2001. Spliceosomal UsnRNP biogenesis,
structure and function. Curr. Opin. Cell Biol. 13:290–301.
48. Yong, J., L. Pellizzoni, and G. Dreyfuss. 2002. Sequence-specific interaction
of U1 snRNA with the SMN complex. EMBO J. 21:1188–1196.
49. Yong, J., L. Wan, and G. Dreyfuss. 2004. Why do cells need an assembly
machine for RNA-protein complexes? Trends Cell Biol. 14:226–232.
VOL. 25, 2005BIOCHEMICAL DEFICIENCY IN SPINAL MUSCULAR ATROPHY 5551