Abstract
Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by homozygous loss of the Survival Motor Neuron 1 (SMN1) gene. In the absence of SMN1, inefficient inclusion of exon 7 in transcripts from the nearly identical SMN2 gene results in ubiquitous SMN decrease but selective motor neuron degeneration. Here we investigated whether cell type-specific differences in the efficiency of exon 7 splicing contribute to the vulnerability of SMA motor neurons. We show that normal motor neurons express markedly lower levels of full-length SMN mRNA from SMN2 than do other cells in the spinal cord. This is due to inefficient exon 7 splicing that is intrinsic to motor neurons under normal conditions. We also find that SMN depletion in mammalian cells decreases exon 7 inclusion through a negative feedback loop affecting the splicing of its own mRNA. This mechanism is active in vivo and further decreases the efficiency of exon 7 inclusion specifically in motor neurons of severe-SMA mice. Consistent with expression of lower levels of full-length SMN, we find that SMN-dependent downstream molecular defects are exacerbated in SMA motor neurons. These findings suggest a mechanism to explain the selective vulnerability of motor neurons to loss of SMN1.
INTRODUCTION
Spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by degeneration of motor neurons (MNs) in the spinal cord and by skeletal muscle atrophy (8, 38, 44). SMA is the most common genetic cause of infant mortality and is classified into three types based on the age of onset and clinical severity (50, 56). Regardless of disease severity, all SMA patients have homozygous deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene—the SMA-determining gene—and retain at least one copy of the nearly identical SMN2 gene (33). While the SMN1 gene produces full-length transcripts, the SMN2 gene mainly produces an alternatively spliced mRNA lacking exon 7 (SMNΔ7). Since the SMNΔ7 protein is unstable and rapidly degraded (32, 36), the low levels of full-length functional SMN produced by the SMN2 gene cannot compensate for the loss of SMN1, resulting in SMA. Thus, SMA is caused by decreased expression of SMN protein, and correspondingly, disease severity correlates well with the degree of SMN reduction in SMA patients (19, 34). Importantly, the SMN2 gene copy number varies in the human population and acts as the principal disease modifier in SMA because the presence of more SMN2 copies generally coincides with a milder clinical outcome (41). Studies of human SMA patients and animal models of disease indicate that low SMN levels from SMN2 are sufficient for normal function of most cells but not motor neurons (8, 44). However, the reason for the selective vulnerability of motor neurons to SMN deficiency is unknown.
Since the efficiency of exon 7 splicing determines the amount of functional SMN produced by SMN2, the mechanisms underlying the alternative splicing of exon 7 in SMN1 and SMN2 mRNAs have been subject to extensive studies. This is for two main reasons: the critical relevance of exon 7 splicing in SMA etiology and its role as a potential target of therapeutic intervention through splicing correction. To date, an array of cis-acting elements and trans-acting factors has been implicated in the regulation of SMN exon 7 splicing (5, 51, 58). Most importantly, a single nucleotide difference between SMN1 and SMN2—a C-to-T transition at position +6 of exon 7—has been found to be largely responsible for the differential inclusion of exon 7 in SMN transcripts (37, 45). Whereas the C in SMN1 exon 7 is thought to form an exonic splicing enhancer (ESE) element that is bound by ASF/SF2 to promote exon inclusion (12, 13), the T at the same position in SMN2 exon 7 not only disrupts the ESE motif but also creates an exonic splicing silencer (ESS) that is bound by hnRNP A1 and Sam68, causing enhanced exon skipping (30, 51). Recent studies identified sequence variants of SMN2 that increase exon 7 inclusion and are associated with reduced disease severity in patients (6, 55, 60), further underscoring the importance of exon 7 splicing in SMA pathology.
The critical role of RNA splicing in SMA etiology is not limited to the regulation of exon 7 inclusion but also involves the function of the SMN protein. SMN and at least eight additional proteins termed Gemin2 to Gemin8 and Unrip form a large multiprotein complex known as the SMN complex (2, 10, 11, 16, 17, 25, 35, 53). The only molecularly defined cellular function of the SMN complex is in the biogenesis of small nuclear ribonucleoproteins (snRNPs) (3, 40, 47, 52), which are the essential components of pre-mRNA splicing machinery. Spliceosomal snRNPs of the Sm class are comprised of one snRNA molecule (U1, U2, U4, U5, U11, U12, and U4atac) and seven common Sm proteins, as well as auxiliary factors specific to each snRNA (61). The activity of the SMN complex is required for the assembly of the heptameric core of Sm proteins on snRNAs that occurs in the cytoplasm (14, 21, 43, 54). Our previous studies in mouse models of SMA have demonstrated a direct correlation between disease severity and the degree of snRNP assembly defects caused by SMN deficiency in the spinal cord (22). Impairment of this essential, SMN-dependent pathway decreases snRNP levels in tissues of severe-SMA mice (22, 64). Importantly, restoration of normal snRNP levels coincides with phenotypic correction in animal models of SMA (62, 63). Thus, increasing evidence links SMN-dependent disruption of snRNP biogenesis to SMA pathology (15, 52). However, the identity of SMN target mRNAs whose defective splicing may contribute to motor neuron dysfunction in SMA remains elusive.
To date, therefore, a dual connection between RNA splicing and SMA etiology has emerged. On one hand, the decrease in SMN protein levels that causes SMA in the absence of SMN1 is due to inefficient splicing of exon 7 in transcripts from SMN2. On the other hand, the function of SMN in the assembly of snRNPs is essential for RNA splicing and impaired in SMA. In this work, we have tested the hypothesis that cell type-specific differences in the efficiency of exon 7 splicing may contribute to the selective vulnerability of motor neurons in SMA. We show that normal motor neurons express markedly lower levels of full-length SMN mRNA from the SMN2 gene than other cell types in the spinal cord and that this is due to particularly inefficient exon 7 inclusion. We also highlight a negative feedback loop whereby SMN-dependent changes in the snRNP abundance decrease exon 7 splicing in mammalian cells, consistent with the conclusions of a recent study (29). Importantly, analysis of severe-SMA mice revealed that this feedback mechanism is active in vivo in motor neurons but not in other spinal cord cells. Consistent with a greater reduction in SMN levels upon loss of SMN1, we provide evidence that downstream molecular defects linked to SMN deficiency are strongly and specifically exacerbated in SMA motor neurons. Our findings identify SMN2 exon 7 inclusion as an SMN-dependent splicing event that is relevant to SMA pathology. They also uncover a plausible mechanism to explain the selective vulnerability of motor neurons in SMA that is based on cell type-specific differences in the efficiency of exon 7 inclusion and their reduced ability to produce functional SMN protein from the SMN2 gene compared to other spinal cells.
MATERIALS AND METHODS
Tissue culture and DNA transfection.
NIH 3T3 cells were grown in Dulbecco modified Eagle medium (DMEM) with high glucose (Gibco) containing 10% fetal bovine serum (HyClone), 2 mM glutamine (Gibco), and 0.1 mg/ml gentamicin (Gibco). RNA interference (RNAi) was induced where needed by addition to the growth medium of doxycycline (Dox; Fisher) at a final concentration of 100 ng/ml, and cells were collected after 5 days. For transient-transfection experiments, 4 × 105 cells were transfected with 4 μg of either SMN1 or SMN2 splicing reporter constructs (30) using PolyFect transfection reagent (Qiagen) according to the manufacturer's instructions. Following incubation for 12 h at 37°C, the growth medium was changed and cells were incubated for an additional 24 h at 37°C prior to collection.
NIH 3T3 cell lines.
The NIH 3T3 cell lines used in this study were generated through lentiviral transduction followed by antibiotic selection and cloning by limiting dilution. With the exception of commercially available pLenti6/TR (Invitrogen)—which constitutively expresses the tetracycline-dependent repressor (TR) protein under the control of the cytomegalovirus (CMV) promoter—all other lentiviral constructs were generated by standard cloning techniques using pRRLSIN.cPPT.PGK-GFP.WPRE as a backbone (Addgene plasmid 12252). Viral stocks pseudotyped with the vesicular stomatitis G protein (VSV-G) were prepared by transient cotransfection of 293T cells using the ViraPower lentiviral packaging mix (Invitrogen) according to the manufacturer's instructions. NIH 3T3-SmnRNAi cells were generated by serial transduction of wild-type NIH 3T3 cells with pLenti6/TR and a lentiviral construct expressing a short hairpin RNA (shRNA) targeting mouse Smn mRNA (5′-GAAGAAUGCCACAACUCCC-3′) under the control of a tetracycline-regulated H1 promoter. NIH 3T3-SMN/SmnRNAi cells were generated by transduction of NIH 3T3-SmnRNAi cells with a lentiviral construct constitutively expressing epitope-tagged human SMN (Flag and Strep fused in tandem at the amino terminus) under the control of the phosphoglycerate kinase (PGK) promoter. NIH 3T3-SmBRNAi cells were generated by serial transduction of wild-type NIH 3T3 cells with pLenti6/TR and a lentiviral construct expressing an shRNA targeting mouse SmB mRNA (5′-GCAAGATGCTGCAGCACAT-3′) under the control of a tetracycline-regulated H1 promoter.
Mouse breeding and tissue collection.
FVB.Cg-Tg(SMN2)89Ahmb Smn1tm1Msd/J (JAX stock no. 005024) mice were interbred to obtain severe-SMA (Smn−/−; SMN2+/+) mice (46). FVB.Cg-Tg(SMN2*delta7)4299Ahmb Tg(SMN2)89Ahmb Smn1tm1Msd/J (JAX stock no. 005025) mice were interbred to obtain Δ7 SMA (Smn−/−; SMN2+/+; SMNΔ7+/+) mice (32). Δ7 SMA mice specifically expressing green fluorescent protein (GFP) in motor neurons were generated by crossing FVB.Cg-Tg(SMN2*delta7)4299Ahmb Tg(SMN2)89Ahmb Smn1tm1Msd/J (JAX stock no. 005025) mice to B6.Cg-Tg(Hlxb9-GFP)1Tmj/J (JAX stock no. 005029) mice. The resulting carrier mice (Smn+/−; SMN2+/+; SMNΔ7+/+; HB9:GFP+/−) were interbred for more than 10 generations to obtain Δ7 SMA (Smn−/−; SMN2+/+; SMNΔ7+/+; HB9:GFP+/−) mice. The phenotype of these mice was no different from that of line 005025. All experiments were conducted in accordance with the protocols described in the National Institutes of Health Guide for the Care and Use of Animals and were approved by the Institutional Laboratory Animal Care and Use Committee of Ohio State University and Columbia University. Spinal cord tissue from control and SMA mice was rapidly dissected and immediately frozen in liquid nitrogen. A tail biopsy specimen was also taken from each mouse and used for genotyping as previously described (22). All tissue samples were stored at −80°C until use.
Immunohistochemistry.
Immunohistochemistry experiments were carried out using normal (Smn+/+; SMN2+/+; SMNΔ7+/+) and Δ7 SMA (Smn−/−; SMN2+/+; SMNΔ7+/+) mice at postnatal day 10. Mice sacrificed by CO2 gas inhalation were transcardially perfused first with phosphate-buffered saline (PBS) and then with 4% paraformaldehyde (PFA)-0.1 M phosphate buffer (PB). Spinal cords were dissected, postfixed for 2 h in 4% PFA-0.1 M PB, cryoprotected for 3 days in 30% sucrose, and then embedded in Tissue-Tek O.C.T. compound (Sakura Finetechnical, Tokyo, Japan). T10 transverse sections (20 μm) of the spinal cord cut on a cryostat were blocked for 30 min in a solution containing 3% bovine serum albumin (BSA), 5% donkey serum, and 1% Triton X-100 and then incubated for 2 days with a mouse monoclonal anti-SmB antibody (18F6; 1:200) and a rabbit polyclonal anti-Hb9 antibody (Abcam; 1:16,000). After several washes, the sections were incubated for 3 h with an Alexa Fluor 488-conjugated donkey anti-mouse IgG and an Alexa Fluor 594-conjugated donkey anti-rabbit IgG (Invitrogen; 1:500) before being mounted in Vectashield (Vector Laboratories, Burlingame, CA). Images were acquired on a laser scanning confocal microscope (Bio-Rad 1024; Bio-Rad, Hercules, CA) under identical exposure and gain settings. Hb9 and SmB levels in the nucleus of motor neurons were quantified from Z-stack confocal images by measuring mean fluorescence intensity per area using ImageJ 1.37v software.
LCM.
Spinal cord sections (L1 to L5) were harvested from control (Smn+/−; SMN2+/+; SMNΔ7+/+; HB9:GFP+/−) and Δ7 SMA (Smn−/−; SMN2+/+; SMNΔ7+/+; HB9:GFP+/−) mice at postnatal day 6, mounted, and flash frozen in liquid nitrogen-cooled isopentane. Spinal cords were cryosectioned (14 μm) and made to adhere to UV-treated PEN membrane slides (Zeiss). Tissue sections were immediately fixed in methanol for 30 s, Nissl stained in 1% cresyl violate acetate for 1 min, and immediately stored at −80°C. Laser capture microdissection (LCM) was performed with the Palm Microbeam (Carl Zeiss MicroImaging) under 10× magnification. Motor neurons were identified by size and position in the ventral horn of the spinal cord. Approximately 500,000 μm2 of motor neuron tissue was collected per sample. For each sample, an additional 500,000 μm2 of non-motor neuron tissue was collected from the dorsal horn of the same spinal cord sections.
RNA purification and RT-PCR analysis.
RNA from NIH 3T3 cells and whole spinal cord tissue was extracted with TRIzol reagent (Invitrogen) and digested with DNase I (Ambion). RNA was reverse transcribed using the RevertAid first-strand cDNA kit (Fermentas). RNA from LCM samples was purified using the RNAqueous-Micro kit (Ambion; AM1931). One round of linear amplification was performed with the MessageAmp II aRNA amplification kit (Ambion; AM1751) according to the manufacturer's instructions. For analysis of exon 7 splicing by semiquantitative radioactive reverse transcription-PCR (RT-PCR), the forward primer was end labeled using T4 polynucleotide kinase (New England BioLabs) and [γ-32P]ATP (Amersham). PCR products were amplified using AmpliTaq Gold DNA polymerase (Roche), resolved on a 6% polyacrylamide-8 M urea gel, and subjected to autoradiography. Quantification of radiolabeled PCR products was carried out using a Typhoon PhosphorImager (Molecular Dynamics). The linear range of amplification was determined by independent PCRs for each experiment. Primers used for radioactive RT-PCR analysis are listed in Table S1 in the supplemental material. For real-time RT-quantitative PCR (RT-qPCR) analysis, qPCRs were carried out using Power SYBR green PCR master mix (Applied Biosystems) in a Realplex4 Mastercycler (Eppendorf). The qPCR primers used in this study are listed in Table S1 in the supplemental material.
Antibodies.
The antibodies used in this study were as follows: anti-SMN clone 8 (BD Transduction Laboratories), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (Chemicon), anti-Gemin8 1F8 (11), anti-hnRNPR/Q 18E4 (Sigma), anti-hnRNPA1 4B10 (Sigma), anti-Sam68 (Santa Cruz), anti-ASF/SF2 (Santa Cruz), anti-Tra2-β1 (26), anti-SRp20 (Santa Cruz), anti-TDP43 (Proteintech Group), anti-SR proteins (Zymed), anti-SmB 18F6 (11), anti-Sm Y12 (Lab Vision), anti-Hb9 (Abcam), antitubulin DM 1A (Sigma), and anti-Flag M2 (Sigma).
Western blotting.
Cells were lysed in SDS sample buffer (2% SDS, 10% glycerol, 5% β-mercaptoethanol, 60 mM Tris-HCl, pH 6.8, bromophenol blue), passed through a 27-gauge needle five times, and sonicated. Protein extracts were quantified using the RC DC protein assay (Bio-Rad). Samples were run on a 12% SDS-PAGE gel and transferred onto a Trans-Blot transfer medium nitrocellulose membrane (Bio-Rad) using a TE77x semidry transfer unit (Hoefer) using 1× Tris-glycine buffer (Bio-Rad) containing 20% methanol. Membranes were stained with 0.1% (wt/vol) Ponceau S and 5% acetic acid, destained in distilled H2O, and blocked for 1 h at room temperature with 5% nonfat dry milk (LabScientific) in PBS containing 0.1% Tween 20 (Acros). Incubation with primary antibody was performed in PBS containing 0.1% Tween 20 for 1 h at room temperature. Membranes were washed 3 times for 10 min with PBS containing 0.1% Tween 20 at room temperature. Incubation with secondary antibodies conjugated to horseradish peroxidase was performed in PBS containing 0.1% Tween 20 for 1 h at room temperature. Membranes were washed 3 times for 10 min with PBS containing 0.1% Tween 20 at room temperature. Chemiluminescence was carried out using a SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) according to the manufacturer's instructions. Signal was detected by autoradiography using Full Speed Blue sensitive medical X-ray film (Ewen Parker X-Ray Corporation).
RNA pulldown.
The SMN1 and SMN2 RNA probes used in this study have been described elsewhere (39). Biotinylated SMN1 and SMN2 RNA probes were transcribed from DNA templates using the MEGAshortscript T7 transcription kit (Ambion) in the presence of 5 mM ATP/CTP/GTP, 3.75 mM UTP, 1.25 mM biotin-UTP, and [α-32P]UTP (3,000 Ci/mmol). After digestion with Turbo DNase I (Ambion), biotinylated RNAs were separated on an 8% polyacrylamide-8 M urea gel and purified from the gel using RNA elution buffer (300 mM sodium acetate, 0.1% SDS, 1 mM EDTA) in the presence of 10% phenol (phenol-chloroform-isoamyl alcohol, 25:24:1) overnight at room temperature. Whole-cell extracts from NIH 3T3 cells were prepared in buffer E (20 mM HEPES-KOH, pH 7.9, 100 mM KCl, 0.2 mM EDTA, 10% glycerol) containing 1 mM dithiothreitol (DTT), complete EDTA-free protease inhibitors (Roche), and PhosStop phosphatase inhibitors (Roche) as previously described (31). Protein concentration was measured using the Bradford assay (Bio-Rad). For each pulldown, 100 μg of whole-cell extract was incubated with 500 ng of biotinylated RNA in buffer E containing 0.5 mM ATP, 20 mM creatine phosphate, 1.6 mM MgCl2, and 1 μl of RNasin Plus RNase inhibitor (Promega) for 30 min at 30°C. After incubation, RNA-protein complexes were isolated with protein G streptavidin Sepharose High Performance beads (GE Healthcare) in a final volume of 1 ml of RSB-100 (10 mM Tris-HCl, pH 7.4, 2.5 mM MgCl2, 100 mM NaCl) containing 0.1% Nonidet P-40 (NP-40; Accurate Chemical and Scientific Corp.) for 2 h at 4°C while rotating. Beads were washed 5 times with 1 ml of RSB-100 containing 0.1% NP-40, and bound proteins were eluted by boiling in SDS-PAGE buffer prior to Western blot analysis.
snRNP analysis.
For in vitro snRNP assembly experiments, cell extracts from NIH 3T3 cells were prepared in ice-cold reconstitution buffer (20 mM HEPES-KOH, pH 7.9, 50 mM KCl, 5 mM MgCl2, 0.2 mM EDTA, 5% glycerol) containing 0.01% NP-40 as previously described (22). Radioactive U1 snRNA was prepared by transcription from a linearized template DNA in the presence of [α-32P]UTP (3,000 Ci/mmol) and m7G cap analogue (New England BioLabs) and then purified from denaturing polyacrylamide gels according to standard procedures. In vitro snRNP assembly assays with freshly prepared NIH 3T3 cell extracts (10 μg, 20 μg, or 40 μg) were carried out in a volume of 20 μl of reconstitution buffer containing 0.01% NP-40, 10,000 cpm of radioactive U1 snRNA, 2.5 mM ATP, and 10 μM Escherichia coli tRNA for 1 h at 30°C. Following addition of heparin and urea to final concentrations of 5 mg/ml and 2 M, respectively, reaction mixtures were analyzed by electrophoresis on 6% polyacrylamide native gels at 4°C and autoradiography (54).
Analysis of steady-state levels of endogenous snRNPs was carried out as previously described (22). Briefly, NIH 3T3 cell extracts (200 μg) were immunoprecipitated with anti-Sm (Y12) antibodies in RSB-500 buffer (500 mM NaCl, 10 mM Tris-HCl, pH 7.4, 2.5 mM MgCl2) containing 0.1% NP-40, EDTA-free protease inhibitor cocktail (Roche), and phosphatase inhibitors (50 mM NaF, 0.2 mM Na3VO4) for 2 h at 4°C. After extensive washing, snRNAs were 3′ end labeled in the presence of [32P]pCp (3,000 Ci/mmol) and T4 RNA ligase (Roche), followed by electrophoresis on 6% polyacrylamide-8 M urea gels and autoradiography. Quantification was carried out using a Typhoon PhosphorImager (Molecular Dynamics).
Immunofluorescence analysis.
NIH 3T3-SmnRNAi cells cultured on glass coverslips in 24-well plates either in the presence or in the absence of doxycycline were washed briefly with PBS, fixed with 4% PFA-PBS for 30 min at room temperature, and permeabilized with 0.5% Triton X-100–PBS for 5 min on ice. Blocking and incubation with both primary and secondary antibodies were performed using 3% BSA-PBS for 1 h at room temperature. Images were collected with a Leica SP5 confocal microscope.
Statistical analysis.
Statistical analysis was carried out using the unpaired Student t test and the Prism 5 (GraphPad) software. P values are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. Unless otherwise stated, all quantification data are plotted as means and standard errors of the means (SEM) from independent experiments.
RESULTS
SMN deficiency impairs exon 7 inclusion in SMN mRNAs.
We sought to investigate the effects of SMN deficiency on splicing of exon 7 using a stable NIH 3T3 cell line (NIH 3T3-SmnRNAi) in which expression of an shRNA against endogenous mouse Smn mRNA is triggered by addition of doxycycline to the culture medium. Western blot analysis showed that treatment of NIH 3T3-SmnRNAi cells with doxycycline for 5 days strongly reduces the levels of the Smn protein (Fig. 1A). To control for nonspecific, off-target effects of shRNA expression, NIH 3T3-SmnRNAi cells were further engineered to constitutively express an epitope-tagged isoform of human SMN that is resistant to RNAi (NIH 3T3-SMN/SmnRNAi). Figure 1A shows that human SMN is expressed at levels comparable to those of endogenous Smn and is not affected by doxycycline-dependent RNAi induction in NIH 3T3-SMN/SmnRNAi cells. Consistent with previous studies (22, 64), immunoprecipitation experiments with anti-Sm antibodies revealed that Smn depletion in NIH 3T3 cells causes a profound alteration in the levels of spliceosomal snRNPs and that this defect is corrected by ectopic expression of human SMN (Fig. 1B; see also Fig. S1 in the supplemental material).
Fig 1.
SMN deficiency impairs exon 7 splicing. (A) NIH 3T3 cell lines with regulated knockdown of mouse Smn. Western blot analysis of NIH 3T3-SmnRNAi and NIH 3T3-SMN/SmnRNAi cells cultured for 5 days without (−) or with (+) doxycycline (Dox). Blots were probed with an antibody that recognizes both mouse and human SMN proteins to monitor knockdown efficiency, with an anti-Flag antibody to detect epitope-tagged human SMN, and with an antibody against tubulin as a loading control. Addition of Dox to the growth medium specifically induces shRNA expression and consequently RNAi-mediated knockdown of endogenous mouse Smn protein. The slower migration of RNAi-resistant human SMN is due to the presence of an epitope tag at the amino terminus as indicated by its detection with anti-Flag antibodies. (B) Smn deficiency decreases the levels of spliceosomal snRNPs in NIH 3T3 cells. Extracts from NIH 3T3-SmnRNAi and NIH 3T3-SMN/SmnRNAi cells cultured for 5 days either without (−) or with (+) Dox were immunoprecipitated with an anti-Sm antibody followed by 3′ end labeling of RNA, denaturing gel electrophoresis, and autoradiography. Known snRNA species are indicated on the right. Note that most U6 snRNAs harbor a 2′,3′-cyclic phosphate end that is not labeled using this assay. Quantification of snRNP levels from independent experiments is shown in Fig. S2 in the supplemental material. (C) SMN deficiency decreases the efficiency of exon 7 inclusion in NIH 3T3 cells. Radioactive RT-PCR analysis of exon 7 splicing following transfection of SMN1 or SMN2 minigene reporters into NIH 3T3-SmnRNAi cells cultured without (−) or with (+) Dox. Arrows point to exon 7-included (FL) and exon 7-skipped (Δ7) SMN mRNAs. (D) Quantification of the efficiency of exon 7 inclusion in NIH 3T3-SmnRNAi. Blue and red bars correspond to results from NIH 3T3 cells cultured without (−) or with (+) Dox, respectively. Values represent means and SEM from independent experiments (n=4; *, P < 0.05; ***, P < 0.001) as in panel C. Data were normalized relative to −Dox set as 1. (E) Expression of human SMN corrects exon 7 splicing defects caused by depletion of endogenous mouse Smn in NIH 3T3 cells. Radioactive RT-PCR analysis of SMN exon 7 splicing following transfection with either SMN1 or SMN2 minigene reporters into NIH 3T3-SMN/SmnRNAi cells cultured without (−) or with (+) Dox. (F) Quantification of the efficiency of exon 7 inclusion in NIH 3T3-SMN/SmnRNAi cells. Blue and red bars correspond to results from NIH 3T3 cells cultured without (−) or with (+) Dox, respectively. Values represent means and SEM from independent experiments (n=3) as in panel E. Data were normalized relative to −Dox set as 1.
To analyze exon 7 splicing in these cells, we used well-established minigene reporters (30) that express the region between exon 6 and exon 8 of either SMN1 or SMN2 genes under the control of the CMV promoter (see Fig. S2A in the supplemental material). RT-PCR analysis of SMN1 and SMN2 minigenes transiently transfected in wild-type NIH 3T3 cells showed the expected alternatively spliced products corresponding to SMN mRNAs that either contain or lack exon 7 (see Fig. S2B). No RT-PCR products were observed following transfection of an empty vector (mock). Quantification of multiple transfection experiments indicates that exon 7 is included in ∼80% of SMN1 mRNAs and ∼6% of SMN2 mRNAs in NIH 3T3 (see Fig. S2C). Thus, transfected NIH 3T3 fibroblasts display a pattern of exon 7 splicing similar to that of endogenous SMN1 and SMN2 genes in human cells.
We then asked whether SMN levels affect splicing of exon 7 in human SMN1 and SMN2 mRNAs. Strikingly, RT-PCR analysis following transient transfection of splicing reporters in NIH 3T3-SmnRNAi cells depleted of endogenous Smn demonstrates that the efficiency of exon 7 inclusion in both SMN1 (without Dox, 83.6% ± 2.7%; with Dox, 55.5% ± 3.8%; P < 0.001) and SMN2 (without Dox, 6.4% ± 1.1%; with Dox, 3.1% ± 0.7%; P < 0.05) mRNAs was strongly reduced compared to the same cells with normal SMN levels (Fig. 1C and D). Importantly, we found no significant differences in exon 7 splicing for both SMN1 (without Dox, 76.2% ± 2%; with Dox, 77.3% ± 0.2%) and SMN2 (without Dox, 5.5% ± 0.1%; with Dox, 5.6% ± 0.2%) mRNAs in NIH 3T3-SMN/SmnRNAi cells with or without endogenous Smn, indicating that expression of human SMN restored the normal pattern of exon 7 splicing (Fig. 1E and F). Therefore, the observed changes in exon 7 splicing are direct and specific consequences of SMN dysfunction. Moreover, since depletion of SMN impairs exon 7 inclusion in both SMN1 and SMN2 mRNAs to similar extents, the SMN-dependent effect on exon 7 splicing appears to be independent of the C-to-T transition. These results demonstrate that deficiency of SMN protein affects the splicing of its own mRNA through a negative feedback loop that decreases inclusion of exon 7.
SMN deficiency does not affect known regulators of exon 7 splicing.
Several trans-acting factors have been implicated in the regulation of SMN exon 7 splicing (5, 51, 58). To investigate the mechanism by which SMN regulates its own splicing, we first tested the possibility that SMN depletion may influence the expression of these known regulators of exon 7 splicing which, in turn, would lead to inhibition of exon 7 inclusion. To do so, we carried out Western blot analysis of NIH 3T3-SmnRNAi cells cultured in the absence or presence of doxycycline for 5 days as well as wild-type NIH 3T3 cells as a control (Fig. 2A; see also Fig. S3 in the supplemental material). Consistent with previous studies (11, 22, 64), the expression of Gemin8—a core component of the SMN complex (11)—was strongly reduced upon SMN depletion, while doxycycline treatment had no effect in wild-type cells. Interestingly, we found that the expression levels of all known regulators of SMN2 exon 7, including several members of the hnRNP and SR protein families, were unchanged in Smn-deficient NIH 3T3 fibroblasts (Fig. 2A; see also Fig. S3).
Fig 2.
SMN deficiency has no effects on trans-acting regulators of exon 7 splicing. (A) Western blot analysis of wild-type (Control) and NIH 3T3-SmnRNAi cells cultured for 5 days without (−) or with (+) Dox. Blots were probed with antibodies against proteins that have been previously implicated in the regulation of SMN exon 7 splicing. Increasing amounts of protein extract from wild-type NIH 3T3 cells (12.5%, 25%, 50%, and 100%, respectively) were loaded on the left side of the blot as a reference for protein quantification. Gemin8 was used as positive control for a protein whose expression is affected by SMN deficiency (11) and GAPDH as a loading control. (B) Schematic representation of the SMN1 and SMN2 RNA probes used in pulldown assays. The probes encompass the last 68 nucleotides of SMN intron 6, the entire exon 7, and the first 25 nucleotides of intron 7. The nucleotide difference between the SMN1 (C) and SMN2 (T) RNAs as well as the splice junctions (GT and AG) and the branch point adenosine (A) are indicated. (C) Biotinylated RNA probes (SMN1 and SMN2) or no RNA probe (mock) were incubated with whole-cell extracts from NIH 3T3-SmnRNAi cells grown in culture for 5 days without (−) or with (+) Dox. RNA-protein complexes were purified on streptavidin agarose, and bound proteins were analyzed by Western blotting with the indicated antibodies. Input represents 10% of the total amount of protein extract used in the assay.
Even though SMN deficiency does not appear to affect the expression of known splicing regulators of exon 7, it is possible that low SMN levels could influence the subcellular localization of these proteins or their association with SMN pre-mRNA. Immunofluorescence analysis showed that the predominantly nuclear localization of trans-acting factors involved in exon 7 splicing regulation does not change in SMN-deficient NIH 3T3 cells (see Fig. S4 in the supplemental material). To investigate potential changes in the association with SMN pre-mRNA, we carried out pulldown experiments from NIH 3T3 extracts using biotinylated SMN1 and SMN2 RNAs comprising SMN exon 7 and a short portion of flanking introns, including splice sites and the branch point adenosine (Fig. 2B). These RNAs were incubated with whole-cell extracts from control and Smn-deficient NIH 3T3-SmnRNAi cells, and following capture on streptavidin beads and extensive washing, bound proteins were analyzed by Western blotting. Known regulators of exon 7 splicing such as Tra2-β1, Sam68, and hnRNP A1 could be specifically isolated, but their binding to either SMN1 or SMN2 RNAs was not affected by SMN deficiency (Fig. 2C). Significant association of other putative trans-acting regulators of exon 7 splicing as well as SMN binding to its own mRNA was not detected (Fig. 2C). Taken together, these results show that the effects of SMN deficiency on exon 7 inclusion are unlikely to be mediated by changes in the expression, subcellular localization, or function of trans-acting splicing factors.
Defective snRNP biogenesis decreases exon 7 inclusion in SMN mRNAs.
SMN has a well-established function in the assembly of spliceosomal snRNPs that are essential for pre-mRNA splicing (15, 52), and SMN deficiency decreases snRNP levels in NIH 3T3 fibroblasts (Fig. 1; see also Fig. S1 in the supplemental material) as well as tissues of SMA mice (22, 64). These observations raise the possibility that altered snRNP levels may mediate the effects of SMN deficiency on exon 7 splicing. To address this, we analyzed the consequences on exon 7 splicing of specifically disrupting the pathway of snRNP biogenesis without perturbing SMN levels. To do so, we generated a stable NIH 3T3 cell line (NIH 3T3-SmBRNAi) with doxycycline-inducible, RNAi-mediated knockdown of SmB—a core snRNP component (61)—using a lentiviral vector expressing an shRNA targeting endogenous mouse SmB mRNA. Western blot analysis of NIH 3T3-SmBRNAi cells demonstrates specific reduction of endogenous SmB upon addition of doxycycline to the medium without any changes in SMN levels (Fig. 3A). Next, we analyzed the effect of SmB deficiency on SMN-dependent snRNP assembly. To do so, we used a well-established in vitro assay that monitors Sm core formation on radioactive U1 snRNA by gel shift (54) with three increasing amounts of extract from either control or SmB-deficient NIH 3T3 cells (Fig. 3B). This analysis demonstrated that SmB deficiency strongly impairs snRNP assembly. Thus, in these cells the same molecular defect triggered by SMN deficiency—disruption of the snRNP biogenesis pathway—is accomplished in the presence of normal SMN expression levels.
Fig 3.
SmB deficiency impairs snRNP assembly and exon 7 splicing. (A) NIH 3T3 cell lines with regulated knockdown of mouse SmB. Western blot analysis of NIH 3T3 wild-type (Control) and NIH 3T3-SmBRNAi cells cultured for 4 days without (−) or with (+) Dox. Blots were probed with antibodies against SmB to monitor knockdown efficiency and tubulin as a loading control. Note that SMN expression is not decreased by SmB deficiency. (B) SmB deficiency impairs snRNP assembly in NIH 3T3 cells. Three increasing amounts of extracts from NIH 3T3-SmBRNAi cells (10 μg, 20 μg, and 40 μg) cultured for 5 days without (−) or with (+) Dox were analyzed by in vitro snRNP assembly with radioactive U1 snRNA. Reaction mixtures were analyzed by gel shift, and known RNA-protein complexes are indicated on the right (top panel) (54). SmB and SMN protein levels in the extracts used for snRNP assembly were analyzed by Western blotting (bottom panels). (C) SmB deficiency decreases the efficiency of exon 7 inclusion in NIH 3T3 cells. Radioactive RT-PCR analysis of exon 7 splicing following transfection of SMN1 or SMN2 minigene reporters into NIH 3T3-SmBRNAi cells cultured without (−) or with (+) Dox. Arrows point to exon 7-included (FL) and exon 7-skipped (Δ7) SMN mRNAs. (D) Quantification of the efficiency of exon 7 inclusion in NIH 3T3-SmBRNAi. Blue and red bars correspond to results from NIH 3T3 cells cultured without (−) or with (+) Dox, respectively. Values represent means and SEM from independent experiments (n=3; **, P < 0.01; ***, P < 0.001) as in panel C. Data were normalized relative to −Dox set as 1.
To test whether dysfunction in snRNP biogenesis affects exon 7 splicing, SMN1 and SMN2 splicing reporters were transfected in control and SmB-deficient NIH 3T3 cells. RT-PCR analysis shows that SmB deficiency causes a strong decrease in the efficiency of exon 7 inclusion in both SMN1 (without Dox, 86.3% ± 2.5%; with Dox, 44.2% ± 2%; P < 0.001) and SMN2 (without Dox, 6% ± 0.3%; with Dox, 2.3% ± 0.4%; P < 0.01) mRNAs (Fig. 3C and D). These results indicate that SmB-dependent impairment of snRNP assembly affects SMN exon 7 splicing in a manner similar to that of SMN deficiency and that the observed changes in exon 7 splicing regulation likely occur through snRNP-dependent mechanisms.
Motor neurons express lower levels of full-length SMN2 mRNA than do dorsal horn cells in the spinal cord.
We next sought to investigate the regulation of SMN exon 7 splicing in vivo in motor neurons from control and SMA mice. To do so, we relied on laser capture microdissection (LCM) of cells from specific areas of the lumbar spinal cord (Fig. 4A). In addition to motor neurons (MNs) in the ventral horn, we collected cells located in the dorsal horn of the same spinal segment as a control. This area of the spinal cord contains a mixture of glia and neurons but is devoid of motor neurons (here collectively referred to as non-MNs). For these studies, we used spinal cords isolated from SMN2-expressing carrier (Smn+/−; SMN2+/+; SMNΔ7+/+; GFP+/−) and Δ7 SMA (Smn−/−; SMN2+/+; SMNΔ7+/+; GFP+/−) mice. In addition to two copies of the human SMN2 gene, these mice contain several transgenic copies of the SMNΔ7 cDNA and express GFP under the control of the motor neuron-specific Hb9 promoter (32, 42). Compared to mice with only two copies of the SMN2 gene, which survive up to 4 days (46), the presence of the SMNΔ7 transgenes extends the life span of SMA mice to 14 days (32). The Δ7 SMA mouse is the best-characterized and most widely used model of SMA for the study of disease mechanisms as well as preclinical therapy development (8, 49).
Fig 4.
Normal motor neurons express remarkably lower levels of full-length SMN2 mRNA than non-motor neurons due to inefficient exon 7 splicing. (A) Schematic representation (top left) and Nissl-stained image (bottom left) of a transverse section of the mouse spinal cord. Nissl-stained images of the ventral horn area of the spinal cord before (top right) and after (bottom right) laser capture microdissection (LCM) of motor neurons are shown. (B) Validation of LCM motor neurons. Real-time RT-qPCR analysis of the expression ofAtp6, ChAT, and GFP mRNAs relative to GAPDH mRNA in LCM motor neurons (MNs) and LCM non-motor neurons (non-MNs) from the spinal cord of normal mice (Smn+/−; SMN2+/+; SMNΔ7+/+; GFP+/−) at postnatal day 6. (C) Normal motor neurons express much less full-length SMN2 mRNA than do non-motor neurons. Real-time RT-qPCR analysis of the expression of total (FL+Δ7; SMN2TOT) and full-length (FL; SMN2FL) SMN2 mRNAs relative to GAPDH mRNA in LCM MNs and LCM non-MNs. (D) Exon 7 inclusion is less efficient in normal motor neurons than in non-motor neurons. Normalized exon 7 inclusion was measured by real-time RT-qPCR analysis of the expression of full-length (SMN2FL) relative to total (SMN2TOT) SMN2 mRNAs in LCM MNs and LCM non-MNs. Values represent means and SEM from independent experiments (n=8; *, P < 0.05; **, P < 0.01; ***, P < 0.001).
First, we designed and experimentally validated a set of primers (SMN2TOT) that would allow us to measure by RT-qPCR the total levels of human SMN mRNAs (both FL and Δ7) specifically transcribed from the SMN2 gene but not those from the SMNΔ7 transgene (see Fig. S5 in the supplemental material). To do so, we took advantage of the fact that the SMNΔ7 transgene lacks the terminal part of the 3′ untranslated region of the human SMN2 gene (see Fig. S5A). This primer pair (SMN2TOT) and a second set of primers (SMN)—complementary to a region in exon 6 shared by both the SMN2 gene and the SMNΔ7 transgene—were used in RT-qPCR experiments with total RNA from the spinal cord of control (Smn+/+; SMN2+/+), SMA (Smn−/−; SMN2+/+), and Δ7 SMA (Smn−/−; SMN2+/+; SMNΔ7+/+) mice. According to the genotype, analysis with primers specific for mouse Smn showed no expression of mouse Smn mRNA in samples from SMA and Δ7 SMA mice (see Fig. S5B). The SMN-specific primer set detected approximately 9-fold more human SMN mRNA in Δ7 SMA mice than in control and SMA mice (see Fig. S5C). The increase in SMN mRNA levels in Δ7 SMA animals is consistent with the presence of several SMNΔ7 transgene copies in these animals. In contrast, SMN2TOT primers detected the same levels of human SMN mRNA in all mice (see Fig. S5D). The fact that higher RNA levels in Δ7 SMA are not detected when using SMN2TOT primers indicates that these primers specifically monitor the mRNA transcribed from the SMN2 gene and not those coming from the SMNΔ7 transgenes. Finally, both SMN and SMN2TOT primer sets are human specific, as they detect similar levels of SMN mRNA in control and SMA mice both in the presence and in the absence of mouse Smn alleles. Thus, we developed an assay that allows us to measure SMN mRNAs transcribed specifically from the SMN2 gene in SMA mice containing high copy numbers of the SMNΔ7 transgene and that will be useful for future studies in Δ7 SMA mice.
Using this assay, we then carried out RT-qPCR analysis of RNA isolated from LCM MNs and LCM non-MNs of the spinal cord of normal mice (Smn+/−; SMN2+/+; SMNΔ7+/+; GFP+/−). Similar levels of mRNA expression in MNs and non-MNs of another housekeeping gene, Atp6, relative to GAPDH support the use of the latter as an appropriate normalizer between different cell populations (Fig. 4B). Importantly, mRNAs for the motor neuron markers ChAT and GFP were expressed at 50- and 150-fold-higher levels in MNs than in non-MNs, respectively (Fig. 4B), demonstrating very strong enrichment of motor neurons by LCM. We then analyzed the expression levels of total (SMN2TOT) and full-length (SMN2FL) SMN mRNAs specifically transcribed from the SMN2 gene in MNs and non-MNs of normal mice. Strikingly, while total SMN2 mRNA levels did not significantly differ between the two cell populations, MNs expressed 75% less full-length SMN2 mRNA (P < 0.01) than did non-MNs from the same lumbar spinal segment (Fig. 4C). To analyze the mechanism for this remarkable difference, we measured the relative efficiency of exon 7 inclusion in MNs and non-MNs from normal mice following normalization to total SMN2 mRNA levels. Figure 4D shows that exon 7 inclusion is 65% less efficient in MNs than in non-MNs (P < 0.05). Altogether, these results indicate that normal motor neurons are remarkably inefficient in their ability to include exon 7 in SMN2 mRNAs and that this splicing difference likely accounts for a 4-fold reduction in the steady-state levels of full-length SMN2 mRNA compared to other cells in the spinal cord.
SMN deficiency impairs exon 7 inclusion in SMA motor neurons.
To determine the effect of SMN deficiency on exon 7 splicing in SMA mice, we carried out RT-qPCR experiments with total RNA from spinal cord as well as LCM MNs and non-MNs from control (Smn+/−; SMN2+/+; SMNΔ7+/+; GFP+/−) and Δ7 SMA (Smn−/−; SMN2+/+; SMNΔ7+/+; GFP+/−) mice. Analysis of the whole spinal cord revealed no differences in the efficiency of SMN2 exon 7 splicing between control and Δ7 SMA animals (Fig. 5A). Similar results were obtained by semiquantitative RT-PCR as well as RT-qPCR analysis of control (Smn+/+; SMN2+/+) and SMA (Smn−/−; SMN2+/+) spinal cords from a different mouse model of SMA (46) lacking the SMNΔ7 transgene (see Fig. S6 in the supplemental material). Thus, SMN deficiency does not affect exon 7 inclusion generally in neural tissue of SMA mice. In contrast, motor neurons in the lumbar spinal cord of Δ7 SMA mice included ∼50% less exon 7 (P < 0.05) than did normal motor neurons (Fig. 5B). Despite greater experimental variability, likely due to the heterogeneity of cells in the dorsal horn region, no significant difference in the efficiency of exon 7 splicing was observed between non-MNs from control mice and those from Δ7 SMA mice (Fig. 5B), which is also consistent with results in whole spinal cord tissue. These results show that SMN protein deficiency decreases SMN2 exon 7 inclusion in motor neurons in vivo and that this effect appears to be specific for the most vulnerable cell type in SMA.
Fig 5.
SMN deficiency impairs exon 7 splicing in SMA motor neurons. Analysis of the relative efficiency of exon 7 inclusion in the whole spinal cord (A) as well as motor neurons (LCM MNs) and non-motor neurons (LCM non-MNs) (B) isolated from control (Smn+/−; SMN2+/+; SMNΔ7+/+; GFP+/−) and Δ7 SMA (Smn−/−; SMN2+/+; SMNΔ7+/+; GFP+/−) mice at postnatal day 6. Normalized exon 7 inclusion was measured by real-time RT-qPCR analysis of the expression of full-length (SMN2FL) relative to total (SMN2TOT) SMN2 mRNAs. Values represent means and SEM from independent experiments (n=8; *, P < 0.05).
Downstream effects of SMN deficiency are exacerbated in SMA motor neurons.
Further reduction in exon 7 inclusion, combined with the already lower levels of full-length SMN2 mRNA under normal conditions, would be expected to exacerbate the downstream effects of SMN deficiency in SMA motor neurons. To investigate this possibility, we sought to measure the degree of SMN-dependent changes in the expression of downstream targets as a readout of SMN dysfunction in different cell types of the spinal cord. We first looked at changes in the levels of spliceosomal snRNPs that are well-established targets of SMN function affected in SMA (22, 64). Reduced nuclear accumulation of Sm proteins has previously been used as a measure of snRNP biogenesis defects in situ in the mouse spinal cord (28). Our immunohistochemical analysis with antibodies against the snRNP core component SmB revealed the expected widespread expression in the spinal cord of normal mice and predominant localization in the nucleus (Fig. 6A). Despite a remarkable difference in tissue size, SmB expression levels appeared overall similar in the spinal cord of control (Smn+/+; SMN2+/+; SMNΔ7+/+) and Δ7 SMA (Smn−/−; SMN2+/+; SMNΔ7+/+) mice (Fig. 6A and B). This is consistent with the relatively modest reduction of snRNP levels in SMA mice (22, 64). However, SmB immunoreactivity of large ventral horn cells appeared strongly decreased in Δ7 SMA mice compared to normal mice, while no differences among other ventral horn cells or dorsal horn cells were evident (Fig. 6A and B). To determine whether these large ventral horn cells were motor neurons as suggested by their location and size, we carried out double labeling experiments with antibodies against SmB and the motor neuron-specific marker Hb9 (1), followed by quantification of expression levels of these proteins in the nucleus of motor neurons (Fig. 6C and D). These experiments showed that SMN deficiency causes an average 40% decrease (P < 0.001) in the levels of SmB expression in SMA motor neurons. This reduction is specific because similar levels of Hb9 expression were found in motor neurons of normal and Δ7 SMA mice.
Fig 6.
SMN deficiency decreases nuclear levels of SmB protein in SMA motor neurons. (A) Immunohistochemistry analysis with anti-SmB antibodies of a spinal cord section from control (Smn+/+; SMN2+/+; SMNΔ7+/+) mouse at postnatal day 10. Higher-magnification images of dorsal and ventral horn regions are shown on the right. Note the strong ubiquitous nuclear SmB immunoreactivity as well as detectable staining of the cytoplasm in large ventral horn cells that are likely motor neurons. Bars, 10 μm. (B) Immunohistochemistry analysis with anti-SmB antibodies of a spinal cord section from Δ7 SMA (Smn−/−; SMN2+/+; SMNΔ7+/+) mouse at postnatal day 10. Higher-magnification images of dorsal and ventral horn regions are shown on the right. There is a reduction in SmB staining of large ventral horn cells from SMA mice compared to control mice. Bars, 10 μm. (C) Analysis by double-label immunofluorescence and confocal microscopy of spinal cord sections from control (Smn+/+; SMN2+/+; SMNΔ7+/+) and Δ7 SMA (Smn−/−; SMN2+/+; SMNΔ7+/+) mice at postnatal day 10 with anti-SmB and anti-Hb9 antibodies. Arrowheads point to SMA motor neurons with strongly reduced SmB levels. Bars, 10 μm. (D) Quantification of SmB and Hb9 expression in the nucleus of motor neurons. Each data point represents the average fluorescence intensity per unit area in individual Hb9+ nuclei from control (n=72) and SMA (n=42) motor neurons measured from confocal images as in panel C. Means and SEM are shown (***, P < 0.001).
Next, we analyzed changes in the expression of the cyclin-dependent kinase inhibitor 1A (Cdkn1a) mRNA—a negative regulator of the cell cycle also known as p21, Cip1, or Waf1. Previous studies have identified upregulation of Cdkn1a mRNA as a specific downstream effect of SMN deficiency that occurs in tissues from human SMA patients and mouse models (4, 20, 48, 64). This event is mediated by an increase in the stability of Cdkn1a mRNA that is dependent on the activity of two RNA-binding proteins—HuD and KSRP (KH-type splicing regulatory protein)—which interact with SMN and are dysregulated in SMA (7, 24, 27, 59). Consistent with previous studies, we found a significant 1.8-fold (P < 0.05) increase in Cdkn1a mRNA levels in the spinal cord of Δ7 SMA mice compared to unaffected controls (Fig. 7A). Strikingly, LCM SMA motor neurons displayed a 6-fold (P < 0.001) increase in Cdkn1a mRNA expression, while the increase in SMA non-MNs (1.7-fold; P < 0.05) is similar to that found in whole spinal cord (Fig. 7B). Altogether, our results provide direct evidence that these downstream defects triggered by SMN deficiency are more prominent in motor neurons than in other spinal cord cells in SMA mice.
Fig 7.
SMN deficiency exacerbates Cdkn1a mRNA upregulation in SMA motor neurons. RT-qPCR analysis of Cdkn1a mRNA expression in whole spinal cord (A) as well as motor neurons (LCM MN) and non-motor neurons (LCM non-MN) (B) isolated from control (Smn+/−; SMN2+/+; SMNΔ7+/+; GFP+/−) and Δ7 SMA (Smn−/−; SMN2+/+; SMNΔ7+/+; GFP+/−) mice at postnatal day 6. Data were normalized to GAPDH, and for each group, Cdkn1a mRNA levels in Δ7 SMA were expressed relative to those in control. Values represent means and SEM from independent experiments (n=8; *, P < 0.05; ***, P < 0.001).
DISCUSSION
The basis for the selective vulnerability of motor neurons to reduced SMN expression has proven elusive despite progress in our basic understanding of SMA biology. To investigate whether cell type-specific features of SMN gene expression contribute to motor neuron vulnerability in SMA, we looked for differences in the regulation of SMN exon 7 splicing in vivo. We show that normal motor neurons express markedly lower levels of full-length SMN mRNA from the SMN2 gene than do other cells in the spinal cord. This is due to the particularly low efficiency of exon 7 splicing that characterizes motor neurons even under normal conditions. Through studies of loss of SMN function in cellular and animal models, we also show that severe SMN deficiency triggers a negative feedback loop leading to decreased exon 7 inclusion. Importantly, this mechanism is active in vivo and further decreases SMN2 exon 7 splicing specifically in SMA motor neurons. Consistent with expression of lower levels of full-length SMN in motor neurons, we provide evidence that downstream molecular defects caused by SMN deficiency are more prominent in motor neurons than in other spinal cells from SMA mice. Based on our data, we propose that one reason that motor neurons are preferentially vulnerable to loss of SMN1 is because they express less functional SMN from the SMN2 gene than do other cell types.
One of the most important findings of our work came from comparison of SMN2 gene expression levels in motor neurons and other cells in the spinal cord of normal mice. We discovered that motor neurons express 4-fold less full-length SMN2 mRNA than do dorsal horn cells from the same spinal segment. This striking difference is due to changes not in SMN2 gene transcription but in the efficiency of exon 7 inclusion in SMN2 mRNAs. Since inclusion of exon 7 is essential for the stability of the SMN protein (9, 18, 36), this would result in a greater reduction of functional SMN in SMA motor neurons. A 4-fold difference in the levels of full-length SMN2 mRNA can be critical for motor neuron biology and underlie dysfunction of this disease-relevant neural type upon loss of SMN1. Consistent with this conclusion, studies in mouse models showed that two copies of the SMN2 gene produce enough full-length SMN for normal function of most cells except motor neurons causing SMA, whereas eight SMN2 gene copies fully rescue the SMA phenotype (46). This is also in agreement with SMN2 gene copy number being the main disease modifier in human SMA patients (8, 44).
Our analysis in SMA mice identified SMN2 exon 7 inclusion as an SMN-dependent splicing event that is affected in motor neurons and might contribute to SMA pathology. Early studies showed that the ratio of SMN2 exon 7 inclusion is high in lymphoblasts from mild SMA patients and low in those from severe SMA patients (23, 26), highlighting an inverse correlation of clinical severity with the efficiency of SMN2 exon 7 inclusion. We therefore investigated the effects of SMN deficiency on the splicing of its own mRNA and links of this feedback circuitry with SMA pathology. We found a 2-fold decrease in the efficiency of SMN2 exon 7 inclusion in SMA motor neurons compared to control motor neurons. In contrast, we found no significant effect of SMN deficiency on exon 7 splicing in either dorsal horn cells or whole spinal cord. Our findings are in contrast to a recent report showing decreased exon 7 inclusion in the spinal cord of SMA mice (29). Although the reason for this discrepancy is unclear, we note that the previous study limited the analysis to tissue from a single animal per genotype (29), while here we analyzed the spinal cord of two distinct mouse models of severe SMA using at least three animals per group and two distinct techniques. Collectively, our findings indicate that SMN deficiency decreases exon 7 inclusion specifically in motor neurons but not generally in the spinal cord of SMA mice, consistent with the presence of a threshold of SMN reduction that needs to be reached to affect exon 7 splicing in vivo. One reason that this threshold might be reached in SMA motor neurons and not in other spinal cells is the lower levels of functional SMN expressed from SMN2 in motor neurons under normal conditions. This situation would bring about a further reduction in the levels of full-length SMN2 mRNA that might enhance the downstream effects of SMN deficiency specifically in SMA motor neurons.
Our results indicate that SMN deficiency decreases exon 7 splicing through a negative feedback loop activated by disruption of SMN function in snRNP biogenesis. We found that SMN depletion caused a strong decrease in exon 7 inclusion in NIH 3T3 cells. However, this effect appears to be independent of the cis-acting elements and trans-acting factors that regulate exon 7 splicing (5, 51, 58). Low SMN levels decreased exon 7 inclusion of both SMN1 and SMN2 mRNAs to similar extents and did not change the expression or subcellular localization of known regulators of exon 7 splicing as well as their ability to associate with SMN pre-mRNA. Conversely, SMN deficiency decreased the levels of spliceosomal snRNPs, and these changes coincide with reduced exon 7 splicing in both NIH 3T3 fibroblasts and SMA motor neurons. Since exon 7 inclusion may be particularly sensitive to SMN-dependent changes in snRNP abundance due to the presence of suboptimal splice site signals flanking exon 7 (39, 57), we investigated the link between impaired snRNP biogenesis and decreased exon 7 splicing. We found that SmB knockdown in NIH 3T3 cells—which disrupted snRNP assembly without changing SMN levels—caused a reduction in exon 7 splicing remarkably similar to that observed when SMN is depleted. Our findings, together with those of a recent study (29), support the conclusion that SMN-dependent effects on exon 7 splicing are mediated by a decrease in snRNP levels and that the negative feedback loop triggered by SMN deficiency is activated by impairment of its critical function in snRNP assembly. This mechanism likely contributes to decrease the efficiency of exon 7 inclusion in SMA motor neurons compared to normal motor neurons.
The particularly low levels of full-length SMN2 mRNA produced by the SMN2 gene would be expected to exacerbate the downstream effects of SMN deficiency in SMA motor neurons compared to other spinal cord cells upon loss of SMN1. Consistent with this, we found that decreased nuclear accumulation of Sm proteins and upregulation of Cdkn1a mRNA levels are more prominent in motor neurons than in other cells in the spinal cord of SMA mice. Thus, using these SMN-dependent events as readouts of the degree of SMN dysfunction in the spinal cord of SMA mice, we provide evidence that molecular defects linked to SMN deficiency are specifically exacerbated in SMA motor neurons.
Based on our findings, we propose a model to explain the long-standing conundrum of the selective vulnerability of motor neurons in SMA (Fig. 8). Normally, the SMN1 gene produces the vast majority of functional SMN and low levels of full-length SMN are produced from the SMN2 gene due to its characteristically poor inclusion of exon 7. Under wild-type conditions, cell type-specific differences in the efficiency of SMN2 exon 7 splicing would have negligible effects on overall SMN levels and thus little physiological relevance. However, the degree of exon 7 inclusion becomes critical upon homozygous loss of the SMN1 gene (or the Smn gene in mouse models), leaving the SMN2 gene as the only source of functional SMN levels in the organism. According to our model, one reason why SMA motor neurons are selectively vulnerable is because the particularly low efficiency of exon 7 inclusion strongly limits their ability to synthesize full-length SMN from the SMN2 gene at levels achieved in other spinal cord cells. This would result in a greater reduction in SMN levels and a more prominent dysfunction of SMN-dependent downstream pathways leading to degeneration and loss of motor neurons.
Fig 8.
Model for the selective vulnerability of motor neurons in SMA. Under normal conditions, the SMN2 gene produces smaller amounts of full-length SMN mRNA and protein in motor neurons (MN) than in non-motor neurons (non-MN) due to more inefficient exon 7 splicing. Upon loss of the protective SMN1 allele, the downstream effects of SMN deficiency in SMA MNs are exacerbated compared to non-MNs due to the lower SMN levels. These include downregulation of nuclear snRNP levels and upregulation of Cdkn1a mRNA expression. Moreover, reduced snRNP levels trigger a negative feedback loop affecting exon 7 splicing that might contribute to further decreasing SMN expression and enhancing downstream defects specifically in SMA MNs.
Supplementary Material
ACKNOWLEDGMENTS
We thank Christopher Henderson and George Mentis for comments and critical reading of the manuscript. We are grateful to James Manley for the kind gift of SMN1 and SMN2 splicing reporter constructs and to Brunhilde Wirth for antibodies against Tra2-β1. We acknowledge the help of Mariangels de Planell Saguer with experiments in Fig. 3.
This work was supported by grants from the Spinal Muscular Atrophy Foundation (to L.P.) and NIH-NINDS R01NS069601 (to L.P.), R01NS057482 (to U.R.M.), and R01NS038650 (to A.H.M.B.). The National Center for Research Resources provided additional funding for LCM work (award no. UL1RR025755 to A.H.M.B.).
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health.
Footnotes
Published ahead of print 28 October 2011
Supplemental material for this article may be found at http://mcb.asm.org/.
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