## 3 Results
### Characterization of hSMN S290 Phospho-Mutant and Phospho-Mimetic
(**H,I**) NSC34 cells and (**J,K**) HEK 293T cells expressing GFP, GFP-tagged hSMN-FL, hSMN S290D and hSMN S290A (**H,I**) 48 h or (**J,K**) 24 h post-transfection. Co-immunoprecipitation of either (**H,I**) endogenous SMN and hSMN constructs or (**J,K**) ZPR1 and hSMN constructs were performed and determined by Western blot analysis. #, unspecific band. (**I**) Ratios of endogenous SMN to hSMN construct protein levels were quantified by densitometry and normalized to geometric mean (mean \(\pm\) SEM, \(n\) = 4, unpaired \(t\)-test, * \(p\) < 0.05). (**J**) After immunoprecipitation (IP), we found a small amount of GFP in the empty vector control that is not visible in the other IP lanes. (**K**) Intensities of GFP-signals of hSMN constructs were quantified by densitometry and normalized to geometric mean (mean \(\pm\) SEM, \(n\) = 4, unpaired \(t\)-test, * \(p\) < 0.05, ****\(p\) < 0.005). HC represents heavy chain, LC represents low chain. (**L**) Representative immunofluorescence of HEK 293T cells expressing GFP-tagged hSMN-FL, hSMN S290D or S290A (\(n\) = 3). Cells were additionally stained for ZPR1. Scale bar represents 10 \(\mu\)m.  
In SMA patients with missense mutations within the C-terminal YG box of SMN, SMN fails to self-associate [45], and interaction of SMN with snRNNPs is inhibited [46]. However, the mechanism of impaired oligomerization or failure in interaction by mutation of the C-terminus remains unclear. Here, we propose phosphorylation as a regulatory tool for self-oligomerization and protein interaction with ZPR1. Since SMN self-oligomerizes at the YG box and C-terminus [45; 47] and zinc finger protein 1 (ZPR1) interacts with the SMN C-terminus recruiting cytoplasmic SMN into nuclear bodies [15; 48], we next analyzed the impact of altered phosphorylation at S290 on these specific C-terminal SMN functions. Hence, immunoprecipitation served as a tool for determining the SMN oligomerization capacity and the interaction of SMN with ZPR1. The wildtype hSMN-FL as well as the mutants hSMN S290A and hSMN S290D all had the opportunity to bind equal amounts of endogenous SMN (Figure 2H). However, immunoprecipitation of wildtype hSMN and hSMN mutants by their GFP-tags revealed an impaired capacity of oligomerization of hSMN S290D with endogenous SMN compared to hSMN-FL (Figure 2H,I). In contrast, hSMN S290A showed no difference in oligomerization with endogenous SMN compared to hSMN-FL or to its corresponding phospho-mimetic hSMN S290D. Taken together, these findings suggest that a phosphorylated state of SMN at S290 diminishes the oligomerization capacity of SMN. The expression of hSMN S290D was concomitantly reduced compared to hSMN-FL and hSMN S290A in input samples (Figure 2J) as shown earlier in NSC34 cells (Figure 2B,C). Additionally, hSMN-FL and hSMN S290A showed a similar input signal in contrast to the corresponding immunoprecipitation: hSMN S290A revealed an increased binding to ZPR1 as its stronger signal indicates compared to hSMN-FL signal (Figure 2J,K). Immunocytochemistry revealed co-localization of either hSMN-FL or hSMN-S290A with ZPR1 in nuclear bodies (Figure 2L). Regarding hSMN S290D, binding to ZPR1 was strongly impaired in contrast to hSMN-FL and to its non-phosphorylatlable mutant S290A (Figure 2J,K) and failed in co-localization with ZPR1 (Figure 2L). Considering (i) the phosphorylation as a dynamic post-translational modification and (ii) the binding domain of ZPR1 at SMN C-terminus, the phosphorylation state at S290 may modulate the interaction of SMN with ZPR1. Nonetheless, the interaction of SMN and ZPR1 may not be exclusively restricted to a steady state of phosphorylation at S290. To explore the in vivo role of the hSMN S290 residue and its involvement in SMN function in motoneurons, we tested the effects of hSMN S290A and S290D on motoneuron survival in a _C. elegans_model of SMA (Figure 3A,B). _smn-1_, the ortholog of human _SMN1_ in _C. elegans_, is ubiquitously expressed and its depletion causes larval arrest [49; 50].