## 3 Results
### Characterization of hSMN S290 Phospho-Mutant and Phospho-Mimetic  
The serine residue at the C-terminus of human SMN (hSMN S290) is conserved in humans, primates and rodents, whereas a serine at position 292 of hSMN is specific to humans and primates (Figure 2A). We therefore focused on hSMN S290 in the subsequent experiments. To characterize the physiological function of this amino acid residue, we constructed a non-phosphorylatable mutant (by replacing serine for alanine; S290A) and a phospho-mimetic (replacing serine by aspartic acid; S290D) of hSMN by site-directed mutagenesis. These constructs and wildtype (WT) hSMN-FL (full length) controls, respectively, were transfected into NSC34 cells and the expression was analyzed 48 h or 72 h post-transfection. Western blot analysis revealed that _hSMN-FL_ and _hSMN S290A_ were expressed at similar levels whereas _hSMN S290D_ showed significantly decreased expression (Figure 2B,C). Since the measurements reflect steady states and we aimed to determine the stability of SMN and mutants quantitatively, we performed radioactive pulse-chase experiments (Figure 2D,E). Decay data were fitted using a one-phase decay model after normalization of t\({}_{0}\) values. Half-life times revealed similar t\({}_{1/2}\) values for hSMN-FL and mutant S290A, whereas S290D showed decrease of t\({}_{1/2}\) to 39% compared to WT control. Therefore, the phospho-mimetic S290D displays an impaired stability, indicating a phosphorylation-dependent effect. In agreement with the expression analysis (Figure 2B), _hSMN S290D_ also showed decreased expression at the zero hour time point compared to the control and _hSMN S290A_ mutant (Figure 2D) on the autoradiographs. To analyze the consequences of impaired stability mediated by phosphorylation of the single amino acid residue S290 in a cellular context, we next studied the number of SMN-positive nuclear bodies (NBs). Indeed, SMN localizes to these structures denoted as Cajal bodies and nuclear gems [42; 43]. Cajal bodies are involved in modification and processing of snRNPs, a structure that becomes assembled by SMN in the cytoplasm before translocation to the nucleus. Phosphorylation at SMN's N-terminus is important for proper assembly of the SMN complex and pre-snRNP formation of the splicing machinery [22; 24; 25]. Thus, low SMN levels decrease snRNPs and numbers of SMN-positive NBs, a phenotypic hallmark of SMA [44]. NSC34 cells were transfected with the constructs coding for GFP-tagged _hSMN-FL_ and the _S290A_ and _S290D_ mutants. SMN-positive NBs were quantified, and frequency distributions were calculated (Figure 2F,G). S290D showed a significantly decreased number of SMN-positive NBs. This indicates impaired stability and/or incorporation in NBs of this phospho-mimetic compared to control and the non-phosphorylatable SMN. Figure 2: Characterization of hSMN S290 phospho-mutant and phospho-mimetic. (**A**) Multiple sequence alignment of the C-termini of SMN from different species showing conservation of serine 290 residue. (**B**,**C**) Expression of hSMN-FL, hSMN S290D and hSMN S290A in NSC34 cells by Western  
blot 48 h or 72 h post-transfection (mean \(\pm\) SEM, \(n\) = 6, one-way ANOVA with Sidak post-test, ****\(p\) < 0.0001). (**D,E**) Radioactive pulse-chase experiment determined stability of hSMN-FL, hSMN S290D and hSMN S290A. Representative autoradiographs at the same exposure times and decay curves of immunoprecipitated hSMN-FL, hSMN S290D, and hSMN S290A (mean \(\pm\) SEM, \(n\) = 3, non-linear regression, one phase decay, R\({}^{2}\)(hSMN-FL) = 0.815, R\({}^{2}\)(hSMN S290D) = 0.854, R\({}^{2}\)(hSMN S290A) = 0.846). (**F**) Representative fluorescence images display NSC34 cells expressing GFP-tagged hSMN-FL, hSMN S290D or S290A. Scale bar represents 20 \(\mu\)m. (**G**) Nuclear bodies (NBs) were quantified in NSC34 cells expressing hSMN-FL, hSMN S290D or hSMN S290A (mean \(\pm\) SEM, \(n\) = 3, one-way ANOVA with Sidak post-test, ****\(p\) < 0.01).