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. 2006 May 31;34(10):2925-32.
doi: 10.1093/nar/gkl374. Print 2006.

Depletion of SMN by RNA interference in HeLa cells induces defects in Cajal body formation

Cyrille Girard  1 , Henry Neel, Edouard Bertrand, Rémy Bordonné
Affiliations

Depletion of SMN by RNA interference in HeLa cells induces defects in Cajal body formation

Cyrille Girard et al. Nucleic Acids Res. .

Abstract

Neuronal degeneration in spinal muscular atrophy (SMA) is caused by reduced expression of the survival of motor neuron (SMN) protein. The SMN protein is ubiquitously expressed and is present both in the cytoplasm and in the nucleus where it localizes in Cajal bodies. The SMN complex plays an essential role for the biogenesis of spliceosomal U-snRNPs. In this article, we have used an RNA interference approach in order to analyse the effects of SMN depletion on snRNP assembly in HeLa cells. Although snRNP profiles are not perturbed in SMN-depleted cells, we found that SMN depletion gives rise to cytoplasmic accumulation of a GFP-SmB reporter protein. We also demonstrate that the SMN protein depletion induces defects in Cajal body formation with coilin being localized in multiple nuclear foci and in nucleolus instead of canonical Cajal bodies. Interestingly, the coilin containing foci do not contain snRNPs but appear to co-localize with U85 scaRNA. Because Cajal bodies represent the location in which snRNPs undergo 2'-O-methylation and pseudouridylation, our results raise the possibility that SMN depletion might give rise to a defect in the snRNA modification process.

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Figures

Figure 1
Figure 1
Depletion of SMN in Hela cells using an RNAi approach (A) western blot analysis on SMN-depleted cells. An siRNA duplex has been transfected into Hela cells for the indicated time, and extracts were prepared and examined for SMN levels by western blotting using anti-SMN antibodies. A control siRNA was introduced into HeLa cells under similar conditions. The GAPDH protein was used as a loading control. (B) Quantification of SMN protein amounts. Blots were scanned and quantified using ImageQuant software (Molecular Dynamics). The percentage of SMN levels with respect to control is shown. (C) Immunofluorescence studies on SMN-depleted and control Hela cells using anti-SMN antibodies. In control cells, the SMN protein localizes to the cytoplasm and to Cajal bodies, while cytoplasmic and Cajal bodies fluorescence are no longer visible in the SMN-depleted cells. The nucleus was stained with DAPI. Note that the cell presenting Cajal bodies staining in the SMN-depleted cells (right panel) has not taken up the siRNA and can be considered as a control cell.
Figure 2
Figure 2
Glycerol gradient sedimentation of snRNPs. Extracts prepared from SMN-depleted (siRNA-SMN) and control cells (control) after 60 h were analyzed by glycerol gradient centrifugation and fractions were recovered. The RNA was extracted, run on denaturing polyacrylamide gels and transferred to a nylon membrane for northern analysis using 32P-labelled oligonucleotide probes specific for U1, U2, U4, U5 and U6 snRNAs. The fraction number and the positions of the different snRNP complexes are shown at the bottom.
Figure 3
Figure 3
Depletion of SMN hinders the efficient nuclear import of a transiently expressed GFP-SmB fusion protein. After transfection of Hela cells with a siRNA duplex specific to SMN or a control siRNA for 36 h, cells were transfected with a plasmid encoding a GFP-SmB gene. After 24 h, cells were fixed and the subcellular localization of the GFP-SmB fusion protein was then observed by fluorescence microscopy. Cajal bodies were visualized by immunofluorescence using anti-coilin antibodies. As shown in the upper panels, in control cells, the GFP fusion protein localizes to the nuclear compartment and in Cajal bodies while in the SMN-depleted cells (lower panels), a cytoplasmic accumulation of the GFB-SmB fusion protein is observed.
Figure 4
Figure 4
SMN-depleted cells contain numerous foci reacting with coilin antibodies. (A) Immunofluorescence studies with antibodies directed against coilin and SMN were performed on control cells and on SMN-depleted cells. In control cells, coilin and SMN localizes to canonical Cajal bodies while these structures become dispersed in SMN-depleted cells where coilin localizes to numerous nucleoplasmic foci. Zooms of representative cells corresponding to the insets are represented at the right. (B) Relocalization of coilin into nucleoli upon SMN depletion. Immunofluorescence studies with coilin allows the detection of Cajal bodies in control cells while fibrillarin is found primarily in nucleoli and also in Cajal bodies (white arrows, panel b). In SMN-depleted cells (lower panels), coilin is found in residual Cajal bodies and in nucleoli (white arrows, panel h) which are stained by fibrillarin (panels f–g). The nucleus was stained with DAPI.
Figure 5
Figure 5
snRNPs do not localize in residual Cajal bodies observed in SMN-depleted cells. To determine the localization of snRNPs, control cells and SMN-depleted cells were subjected to immunofluorescence studies using anti-p80 coilin (red) and anti-m3G antibodies (green). In control cells, snRNPs accumulate into Cajal bodies and into speckles while in SMN-depleted cells, snRNPs are not located into residual Cajal bodies but are found in a diffuse speckled distribution.
Figure 6
Figure 6
In situ localization of U85 scaRNA. A plasmid containing human U85 scaRNA was transfected into HeLa cells with control or SMN specific siRNA duplexes and after 48 h, cells were subjected to in situ hybridization using a Cy3-fluorescent probe specific to U85 (red). The canonical Cajal bodies in control cells and the residual Cajal bodies in SMN-depleted cells were visualized with anti-p80 coilin antibody staining (green).
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References

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