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Fig. 2. Systematic analysis of protein domains in subcellular distribution of SMN in transfected cells. (A) SMN is depicted with the K-rich, Tudor, P-rich, YG-box and ex7-encoded domains. SMN and deletion mutants were fused to the N-terminus of eGFP (FP). Overexpressed FPs localized in the nucleus within nucleoli (No), the nucleoplasm (Np) and/or CBs; +, presence and -, absence of protein. (B) Immunoblot analyses of lysates from COS cells transiently transfected with the indicated constructs with anti-GFP antibodies revealed that SMN, SMNex7, SMNC40, SMNN86, SMNN194, SMN4725, SMNN86, SMNTudor, SMNex2B recombinants and eGFP showed a major band of apparent mobility of 70, 63, 61, 57, 56, 55, 43, 39, 36 and 28 kDa, respectively. The -SMN and -tubulin incubations served as loading control and relative expression levels, respectively. The position of molecular weight standards is indicated. (C) Distribution pattern of the indicated constructs transiently overexpressed in COS cells and analysed by fluorescence microscopy. Some FPs accumulated in NBs (arrows) and/or in nucleoli (arrow heads). (D) SMN contains a conserved interspecies K-rich sequence (boxed) (Bertrandy et al., 1999). This motif resembles the NoLS identified in proteins, such as MDM2, ARF and coilin (Hebert et al., 2000), and corresponds to a NoLS consensus sequence (Horke et al., 2004). Mutagenesis of the K-rich sequence identified a cryptic NoLS in the N-terminal part of SMN (SMNN86M2) and revealed a role in subnuclear localization of SMN4725 (SMN4725M2) in CBs and nucleoli. Bars, 3 µm.

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