Figure 5.

Genetic and biophysical characterization of Smn YG box mutations in Drosophila. (A) Cartoon of the dmSMN protein, showing the conserved YG box, Gemin2 binding (Gem2) and Tudor domains, along with the location of the 3x-FLAG tag used for transgenic rescue experiments. An alignment of YG box sequences from the human (H.sapiens), zebrafish (D.rerio), butterfly (P.xuthus) and fruitfly (D.melanogaster) SMN orthologs is shown for reference. Phenotypic comparisons of an extensive panel of Drosophila YG box substitution mutations are summarized below the alignment. With the exception of S201F and G202S, which are point mutations in the endogenous Smn gene (55), the rest of the panel is comprised of transgenic constructs that have been recombined with an Smn null allele ((21); this work). Genetic complementation analysis (Comp) was performed using either a wild-type (WT) or mutant Flag-Smn transgene. Mutants that correspond to human SMA-causing missense alleles are shown in bold text; the classification system used for fly SMA models was described previously (44). See Supplementary Figure S4 for pupal and adult viability analysis of the fifteen new transgenic fly lines used in this work. Mutant lines that eclose at low frequency (adult escapers) are considered semi-viable; those marked with a # sign are incapable of establishing an independent breeding stock. Class 2^ denotes animals that pupate but display a significant advancement in lethal phase or reduction in eclosion frequency. The asterisk * = stop codon. (B) SEC-MALS analysis of SMN•G2 complexes containing mutations at hsH273 (H273R) or dmY204 (Y204H and Y204R) aimed at modeling an SMA-causing missense allele in human SMN1. (C) Analytical ultracentrifugation of Drosophila SMN•G2 complexes. c(S) sedimentation distributions of wild-type (WT) or mutant (Y204H and Y204R) are shown. Note, biophysical analyses of additional Drosophila SMN constructs are presented in Table 1 and Supplementary Figure S2.