Deletion and mutation in the SMN Tudor domain specifically impair SMN-HuD interaction. A. Schematic representation of SMN deletion constructs lacking exon 7 (SMNΔ7), the N-terminus (SMNΔN53), or the Tudor domain (SMNΔT). The E134K (SMNE134K) or the G279V (SMNG279V) mutations were introduced in the murine full-length SMN (SMN FL) sequence. B–C. Immunoprecipitation (IP) experiments with HEK293 cells transfected with EGFP- and FLAG-tagged SMN and HuD constructs. Anti-FLAG antibody was used to precipitate FLAG-tagged HuD. Monoclonal antibodies to GFP and FLAG were used for detection. SMN with the Tudor domain deletion (SMNΔT) failed to co-precipitate with HuD (B, lane 7), in contrast to SMN full-length and N- or C-terminal deletions. The Tudor domain alone, fused to EGFP, is sufficient to be co-purified with FLAG-HuD (C, lane 5). P, IP pellet; S, IP supernatant. D–E. BiFC was used to investigate the effect of Tudor domain deletion or mutation on SMN-HuD interaction (D) and SMN self association (E). Neuro2a cells were transfected with BiFC constructs and fixed after 12 hours. Murine full-length SMN (SMN FL), SMNΔT, and Tudor domain (SMNE134K) or YG-box mutants (SMNG279V) were compared. Fluorescence intensity of the BiFC signal (green) was quantified and normalized to CFP protein expression (blue). Both deletion (SMNΔT) and mutation (SMNE134K) in the Tudor domain, but not in the YG-box (SMNG279V), showed significantly reduced interactions with HuD (D, graph). In contrast, SMNG279V mutant showed impaired ability to self-associate compared to both SMN FL and the SMNE134K constructs (E, graph). Scatter plots represent the quantification of 80 to 120 cells per condition from three independent experiments. Mean and SEM are shown (one-way ANOVA and Tukey’s post-hoc test, **p<0.01, ***p<0.001). F. Primary motor neurons were transfected with EGFP-fusions of wild type or mutant SMN cDNAs (SMN FL, SMNE134K, and SMNG279V respectively; green), and stained with tau antibody (red) to recognize the axons. EGFP fluorescence intensity was evaluated in the proximal axonal segment. A significant reduction was observed when the G279V mutation was present (one-way ANOVA and Tukey’s post-hoc test, n=21, 24 and 27 for SMN FL, SMNE134K, and SMNG279V respectively, *p<0.05). Size bars: 10 µm in D, E, and F.