Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCFSlmb degron

Abstract

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1 Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCF^Slmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCF^Slmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCF^Slmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7^S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers.

FIGURE 1:

Flag-SMN immunopurified lysates contain known protein interaction partners and ubiquitin proteasome system (UPS) proteins. (A) Lysates from Oregon-R control (Ctrl) Drosophila embryos and embryos expressing only transgenic Flag-SMN (SMN) were Flag-immunopurified, and protein eluates were separated by gel electrophoresis and silver stained. Band identities predicted by size using information from panels C and D. (B) Direct mass spectrometric analysis of the eluates (which were not gel purified) identified a total of 396 proteins, 114 of which were detected only in SMN sample and 279 of which were detected in both SMN and Ctrl samples. (C) Flag-purified eluates were analyzed by “label-free” mass spectrometry. Numerous proteins that copurify with Flag-SMN are part of the ubiquitin proteasome system (UPS). Of these UPS proteins, Cullin 1 (Cul 1), SkpA, and supernumerary limbs (Slmb) were highly enriched (at least 10-fold) in the SMN sample as compared with Ctrl. (D) Western blot analysis of Flag-purified eluates was used to further validate the presence or absence of SMN interaction partners. Flag-SMN was successfully purified from SMN embryos but was undetectable in the control. As positive controls for known protein interaction partners of SMN, SmB and SmD3 were also easily detectable by Western blotting using anti-Sm antibodies. The presence of Slmb was verified using anti-Slmb. α-Actinin and tubulin were not enriched in our purification and are used as negative controls to demonstrate specificity.

FIGURE 2:

Conserved interaction between SMN and the SCF^Slmb/B-TrCP E3 ubiquitin ligase results in ubiquitylation of SMN. (A) E3 ligases work with E1 and E2 proteins to ubiquitylate their targets. The SCF^Slmb/B-TrCP E3 ubiquitin ligase is made up of three proteins: Cul1, SkpA, and Slmb. The E3 ubiquitin ligase is the substrate recognition component of the ubiquitin proteasome system. (B) Following Cul1-Flag, SkpA-Flag, Flag-Slmb, and Flag-Gem2 immunoprecipitation from Drosophila S2 cell lysates, Western analysis using anti-SMN antibody for endogenous SMN was carried out. Copurification of each of the SCF components with endogenous SMN was detected. (C) An in vitro binding assay tested direct interaction between human SMNΔ5-Gemin2 (SMN•Gem2) (Martin et al., 2012; Gupta et al., 2015) and purified GST-tagged proteins. SMN•Gem2 did not interact with GST protein alone but bound to GST tagged Drosophila SMN (GST-SMN) and GST tagged human B-TrCP1 (GST-B-TrCP1). Levels of GST alone, GST-SMN, and GST-B-TrCP1 were detected using anti-GST antibody. (D) The interaction of Flag-tagged Drosophila SCF components with endogenous human SMN was tested in in HEK 293T cells. Human SMN was detected at high levels following immunoprecipitation of Drosophila Flag-Cul1 and Flag-Slmb and detected at a lower level following Drosophila Flag-SkpA immunoprecipitation. (E) Flag-tagged versions of the human homologues of Slmb, Flag-B-TrCP1, and Flag-B-TrCP2, interact with endogenous human SMN in HEK 293T cells demonstrated by Flag-immunopurification followed by immunodetection of SMN. (F) Protein lysate from HEK 293T cells transfected with 6xHis-Flag-ubiquitin (6HF-Ub) and GFP-SMN was purified using a Ni²⁺ pull down for the tagged ubiquitin. Baseline levels of ubiquitylated GFP-SMN were detected using anti-GFP antibody. Following transfection of Flag-B-TrCP1 or Flag-B-TrCP2, the levels of ubiquitylated SMN markedly increased. Ubiquitylation levels were further increased following addition of both proteins together. In the input, GFP-SMN was detected using anti-GFP antibody, Flag-B-TrCP1 and Flag-B-TrCP2 were detected using anti-Flag antibody, and GAPDH was detected by anti-GAPDH antibody. In the Ni²⁺ pull down, ubiquitylated GFP-SMN was detected using anti-GFP antibody and 6HF-Ub was detected using anti-Flag antibody to verify successful pull down of tagged ubiquitin.

FIGURE 3:

Depletion of Slmb/B-TrCP results in an increase of SMN levels. (A) Depletion of Slmb using 10-d (10d) treatment with dsRNA in Drosophila S2 cells resulted in modestly increased SMN levels. Following Slmb RNAi, full-length SMN levels were increased as compared with cells treated with control dsRNA against Gaussia Luciferase, which is not expressed in S2 cells. (B) The effect of B-TrCP depletion on SMN levels in human cells was tested using siRNA that targets both B-TrCP1 and B-TrCP2 in HeLa cells. We detected a modest increase in levels of full-length endogenous SMN after B-TrCP RNAi but not control (scramble) RNAi. (C) Drosophila S2 cells were treated with cycloheximide (CHX), an inhibitor of protein synthesis, following Slmb depletion following a 3-d dsRNA treatment to test whether differences in protein levels would be exacerbated when the production of new protein was prevented. SMN protein levels were also directly targeted using dsRNA against Smn as a positive control for the RNAi treatment. As a negative control (Ctrl), dsRNA against oskar, which is not expressed in S2 cells, was used. Protein was collected at 0, 2, and 6 h post–CHX treatment. At 6 h post–CHX treatment, there is a modest increase in full-length SMN levels following Slmb RNAi as compared with the initial time point (0 h) and as compared with control RNAi treatment.

FIGURE 4:

Identification and mutation of a putative Slmb/B-TrCP phosphodegron (A) Identification of a conserved putative Slmb phosphodegron (DpSGXXpS/T motif variant) in the C-terminal self-oligomerization domain (YG Box) of SMN. The amino acid sequence of SMN from a variety of vertebrates is shown to illustrate conservation of this motif and rationale for the amino acid changes. Full-length human SMN is labeled as “Human,” and the truncated isoform is labeled “hSMNΔ7.” Endogenous D. melanogaster SMN is labeled “Fruitfly.” To generate a more vertebrate-like SMN, key amino acids in Drosophila SMN were changed to amino acids conserved in vertebrates. Using this SMN backbone, a serine-to-alanine mutation was made in the putative degron in both full-length (vSMN^S201A) and truncated SMNΔ7 (vSMNΔ7A). An additional SMN construct that is the same length as SMNΔ7, but has the amino acid sequence GLRQ (the next amino acids in the sequence) rather than EMLA (the amino acids introduced by mis-splicing of SMN2), was generated. The same serine to alanine mutation was made in this construct as well (MGLRQ* and MGLRQ*^S201A). Finally, to mimic a phosphorylated serine, a full-length vSmn^S201D and truncated vSmnΔ7D were also employed. (B) Western blotting was used to determine protein levels of each of these SMN constructs, with expression driven by the endogenous promoter, in Drosophila S2 cells. Both the vSMN and vSMNΔ7S proteins show increased levels when the serine is mutated to an alanine, indicating disruption of the normal degradation of SMN. Additionally, MGLRQ* protein is present at higher levels than is vSMNΔ7S and protein levels do not change when the serine is mutated to an alanine. Normalized fold change as compared with vSmn levels is indicated at the bottom. *p < 0.05, **p < 0.01, n = 3. (C) Flag-tagged SMN constructs were cotransfected with Myc-Slmb in Drosophila S2 cells. Protein lysates were Flag-immunoprecipitated and probed with anti-Myc antibody to detect SMN-Slmb interaction. In both full-length SMN (vSMN) and truncated SMN (vSMNΔ7), serine-to-alanine mutation decreased interaction of Slmb with SMN. Truncated SMN (vSMNΔ7) showed a dramatically increased interaction with Slmb as compared with full-length SMN (vSMN), despite the fact it is present at lower levels. (D) Full-length SMN constructs containing point mutations known to decrease self-oligomerization (Smn^Y203C and Smn^G206S) and a mutation that does not disrupt self-oligomerization in the fly (Smn^G210V) with and without the serine-to-alanine mutation were transfected in Drosophila S2 cells. The constructs containing the serine to alanine mutation are as follows: Smn^Y203C→Smn^3C-1A, Smn^G206S→Smn^6S-1A, Smn^G210V→Smn^10V-1A. The serine to alanine mutation has a stabilizing effect on SMN mutants with poor self-oligomerization capability. *p < 0.05, n = 3.

FIGURE 5:

Mutation of the Slmb degron rescues defects in SMA model flies. (A) Viability analysis of an SMA point mutation (G206S) in the presence and absence of the degron mutation, S201A. Flies with the following genotypes were analyzed in this experiment: Oregon-R (Ctrl), Flag-Smn^WT,Smn^X7/Smn^X7 (Smn^WT), Flag-Smn^G206S,Smn^X7/Smn^X7 (Smn^G206S), Flag-Smn^G206,S201A,Smn^X7/Smn^X7 (Smn^6S-1A), or Smn^X7/Smn^X7 (Smn^X7). The data for each genotype are expressed as a fraction of pupae or adults over the total number of starting larvae, n = 200. Expression of the WT transgene (Smn^WT) shows robust rescue of the null (Smn^X7) phenotype (∼68% adults). Smn^G206S is a larval lethal mutation. In two independent recombinant lines of Smn^6S-1A (Smn^6S-1A1 and Smn^6S-1A2) a fraction of the larvae complete development to become adults. (B) Locomotor ability of early third-instar larvae was determined by tracking their movement for 1 min and then calculating the velocity. To account for potential differences in larval size, speed is expressed as average body lengths per second moved. Genotypes are as in panel A. Smn^G206S larvae move similarly to null animals. The motility of Smn^6S-1A1 and Smn^6S-1A2 larvae is not different from Ctrl or Smn^WT larvae. ***p < 0.001, n = 50–60 larvae. (C) Larval protein levels were examined by Western blotting; genotypes as in panel A. Lysates from hemizygous mutant lines were probed with anti-Flag or anti-SMN antibodies as indicated. The slower-migrating bands represent the Flag-tagged transgenic proteins and the faster migrating band corresponds to endogenous SMN, which is present only in the Ctrl (note Oregon-R has two copies Smn, whereas the transgenics have only one). Smn^G206S has very low levels of SMN protein. Flies bearing the S201A degron mutation in addition to G206S (Smn^6S-1A) express markedly increased levels of SMN protein.

FIGURE 6:

Stabilization of endogenous SMN and SMNΔ7 in cultured human cells. (A) HEK 293T cells were transfected with equivalent amounts of GFP-SMN∆7A or -SMN∆7S. The following day, cells were harvested after treatment with cycloheximide (CHX) for zero to 10 h. Western blotting with anti-SMN showed that SMN∆7S stabilizes endogenous SMN and SMN∆7 to a greater extent than SMN∆7A. By comparing band intensities within a given lane, we generated average intensity ratios for each time point using replicate blots. We then calculated a “stabilization factor” by taking a ratio of these two ratios. The protective benefit of overexpressing ∆7S vs. ∆7A at t = 0 h was roughly 3.0× for endogenous SMN∆7 and 1.75× for full-length SMN. (B) SMNΔ7A (S270A) expression protects SMA iPSC-derived motor neurons. Control motor neurons were left untreated or transduced with a lentiviral vector expressing an mCherry control. SMA motor neurons were left untreated or transduced with a lentiviral vector expressing an mCherry control or a lentiviral vector expressing SMNΔ7A (S270A). At 4 wk of differentiation, there was no difference in motor neuron survival between control and SMA iPSC motor neuron cultures in any of the treatment groups. However, at 6 wk, SMI-32-positive motor neurons showed selective loss in SMA iPSC motor neuron cultures in the untreated and lenti-mCherry groups compared with control iPSC motor neuron cultures. In contrast, lenti-SMNΔ7A expression fully protects SMA iPSC-derived motor neurons. Representative images of control and SMA iPSC-derived motor neurons labeled with SMI-32 (green) and mCherry (red). Nuclei are stained with 4’,6-diamidino-2-phenylindole (DAPI) and shown in blue. *p < 0.05 by ANOVA. NS = not significant. n = 3

FIGURE 7:

SMN∆7A is a protective modifier of intermediate SMA phenotypes in mice. (A) Mouse genotypes include control unaffected Smn^2B/+ mice, which have a wild-type Smn allele, Smn^2B/– (2B/–) mice treated with scAAV9 expressing different versions of SMN, and untreated 2B/– mice, which are an intermediate mouse model of SMA. 1e11 denotes the viral dose. scAAV9-SMN expresses full-length SMN, scAAV9-SMNΔ7 expresses truncated SMN, scAAV9-SMNΔ7^S270A expresses truncated SMN with the S-to-A change in the degron, and scAAV9-SMNΔ7^S270D expresses truncated SMN with a phosphomimic in the degron. Delivery of AAV9-SMNΔ7A at P1 significantly extended survival in the intermediate 2B/– animals, resulting in 100% of the treated pups living beyond 100 d, similar to the results obtained with the full-length AAV9-SMN construct. Untreated 2B/– animals lived, on average, only 30 d. Mice treated with AAV9-SMNΔ7S survived an average of 45 d. Mice treated with AAV9 expressing SMNΔ7D had an average life span equivalent or slightly worse than that of untreated 2B/– mice. (B) Average weight (measured over time) of the animals used in panel A. AAV9-SMNΔ7A treated mice also gained significantly more weight than either untreated or AAV-SMNΔ7S-treated animals, nearly achieving the same weight as 2B/– pups treated with full-length SMN cDNA. (C) Mouse genotypes include control unaffected Smn^2B/+ mice, which carry a wild-type Smn allele, and 2B/– mice treated with scAAV9 expressing different versions of SMN. scAAV9-SMN expresses full-length SMN and scAAV9-SMNΔ7^S270A expresses truncated SMN with the S-to-A change in the degron. AAV-SMNΔ7A-treated animals retained their improved strength and gross motor functions at late time points (P100), as measured by their ability to splay their legs and maintain a hanging position using a modified tube test.

FIGURE 8:

Proposed model of SMN as a substrate of SCF^Slmb E3 ubiquitin ligase. Unstable SMN monomers, such as those created in SMNΔ7, are the primary substrates for degradation. Active oligomers of full-length SMN (SMN-FL) and partially active SMN-FL•SMN∆7 dimers (Praveen et al., 2014; Gupta et al., 2015) would be targeted to a lesser extent. SCF^Slmb is a multicomponent E3 ubiquitin ligase composed of Slmb, SkpA, Cul1, and Roc1 (see the text for details). This E3 ligase complex functions together with E1 and E2 proteins in the ubiquitin proteasome system (UPS) to tag proteins for degradation by linkage to ubiquitin (Ub). Phosphorylation (P) by GSK3β and/or another kinase (see the text) is predicted to trigger ubiquitylation.