QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 31 January 2006
doi: 10.1242/jcs.02782

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Renvoisé, B. Articles by Lefebvre, S. Search for Related Content PubMed PubMed Citation Articles by Renvoisé, B. Articles by Lefebvre, S.

Distinct domains of the spinal muscular atrophy protein SMN are required for targeting to Cajal bodies in mammalian cells

¹ Laboratoire de Biologie Cellulaire des Membranes, Institut Jacques Monod (IJM), UMR 7592 CNRS/Universités Paris 6 et 7, 2 Place Jussieu, 75251 Paris Cedex 05, France
² Service de Cytométrie, IJM 75251 Cedex 05 Paris, France
³ Laboratoire de Morphométrie et Modélisation Cellulaire, IJM, 75251 Cedex 05 Paris, France
⁴ UR393 INSERM, IRNEM Institute, Hôpital Necker-Enfants Malades, Paris, France

View larger version (99K): [in a new window]   Fig. 1. Localization of endogenous SMN and of FP-SMN in transfected cells. In both (A,B) COS cells and (D,E) type-I-SMA fibroblasts, immunolocalization of the (A,D) endogenous SMN and direct detection of (B,E) FP-SMN revealed that the protein distributes to cytoplasm and nucleoplasm, and concentrates in CBs. (C,F) Enhanced green fluorescent protein (eGFP) diffusely localized throughout the cells. (G) COS cells were double-labelled for SMN (green) and CB marker coilin, gem marker gemin2 (H) and snRNP marker TMG (I) (all red). Inserts show SMN colocalized with coilin, gemin2 and snRNP in CBs. Bars, 3 µm.

View larger version (70K): [in a new window]   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.

View larger version (109K): [in a new window]   Fig. 3. Comparison of localization of SMN proteins using the CB marker coilin. The constructs indicated were transiently transfected into COS cells. Overexpression of FP-SMN, SMNex7, SMN4725 and SMNN86 colocalized with coilin in CBs (red), whereas SMNN86, SMNex2B, SMNTudor, SMNC40 and SMNN189 did not form nuclear foci, and were excluded from CBs. Mutants SMNN86 and SMNex2B accumulated also in nucleoli (see Fig. S1). The microscope was focused on coilin-positive CBs. Inserts show staining for FPs (left) and coilin (middle), and merged images (right). Bars, 3 µm.

View larger version (52K): [in a new window]   Fig. 4. Analysis of protein domains required for nuclear localization of SMN. (A) The proportion of fluorescence in the nucleus relative to the total fluorescence contained in the entire stack of z-sections is presented to visualize the distribution of ten cells for each construct. (B) The 14.3.3 protein localized to the cytoplasm. (C) 14.3.3 fused to the SMN ex2B region remained in the cytoplasm, whereas (D) fusion to the SMN Tudor domain led to accumulation of the protein in the nucleus. Bars, 3 µm.

View larger version (60K): [in a new window]   Fig. 5. Accumulation of endogenous SMN, coilin and snRNPs in CBs of COS cells transiently transfected with SMN constructs. (A) Distribution of snRNPs in cells transfected with SMN mutants: SMN, SMNN86, SMNex7 and SMNTudor (all green) were co-stained with anti-TMG snRNA antibodies (red). (B) Cells transfected with SMN mutants (green) were immunolabelled for endogenous SMN (red). (C) Distribution of coilin, TMG and U2 snRNP-specific B" protein in COS cells transfected with SMNE134K mutant. (D) Distribution of the U2 snRNP-specific B" protein in cells transiently transfected with SMN mutants. Inserts show magnified images of the distribution of those components in NBs of transfected cells. (E) Analyses presented in histograms. Bars, 3 µm.

View larger version (38K): [in a new window]   Fig. 6. Analyses of the interaction of the fluorescently tagged SMN mutants with endogenous SMN and snRNPs. (A) COS cells were transfected with FP-SMN or FP-SMNex7. Immunoblotting shows no significant degradation of gem/CB components in whole-cell extracts prepared from FACS-selected cells. (B-H) Cell extracts were immunoprecipitated with anti-GFP (GFP IP) or anti-TMG snRNA antibodies (TMG IP) and immunoblotted for endogenous SMN. Incubation with anti-GFP served as loading control. SMN mutants coimmunoprecipated the endogenous SMN, except for the Tudor mutant. Both FP-SMN and FP-SMNex7 coimmunoprecipitated with anti-TMG antibodies as confirmed with control IgG (DAKO IP). Light (lig) and heavy chains of immunoglobulins (hig).

View larger version (70K): [in a new window]   Fig. 7. Subnuclear localization of coilin, SMN and TMG of snRNA in SMA-derived fibroblasts. (A) Immortalized control and type I SMA patient fibroblasts were co-stained for SMN (green) and the snRNP marker TMG (red). SMN foci in control cells colocalized with snRNPs, as shown in the insert. By contrast, SMN foci detected in SMA cells were never found snRNP-positive (see insert). It should be noticed that coarse TMG labelling in the nucleoplasm of SMA cells was also observed in controls. (B) Primary fibroblasts derived from control and type I, II and III SMA were immunostained for SMN (green) and coilin (red). (C) Primary fibroblasts derived from control and type I, II and III SMA were immunostained for SMN (green) and TMG (red). Inserts show staining for SMN (left) and coilin or TMG (middle), and the merged image (right). Bars, 3 µm.