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. 2009 Apr 1;18(7):1181-9.
doi: 10.1093/hmg/ddp009. Epub 2009 Jan 7.

The loss of the snoRNP chaperone Nopp140 from Cajal bodies of patient fibroblasts correlates with the severity of spinal muscular atrophy

Benoît Renvoisé  1 , Sabrina Colasse, Philippe Burlet, Louis Viollet, U Thomas Meier, Suzie Lefebvre
Affiliations

The loss of the snoRNP chaperone Nopp140 from Cajal bodies of patient fibroblasts correlates with the severity of spinal muscular atrophy

Benoît Renvoisé et al. Hum Mol Genet. .

Abstract

Spinal muscular atrophy (SMA) is a common autosomal recessive neurodegenerative disease caused by reduced survival motor neuron (SMN) levels. The assembly machinery containing SMN is implicated in the biogenesis of the spliceosomal small nuclear ribonucleoproteins (snRNPs). SMN is present in both the cytoplasm and nucleus, where it transiently accumulates in subnuclear domains named Cajal bodies (CBs) and functions in the maturation of snRNPs and small nucleolar (sno)RNPs. The impact of lowering SMN levels on the composition of CBs in SMA cells is still not completely understood. Here, we analyse the CB composition in immortalized and primary fibroblasts from SMA patients. We show that the U snRNA export factors PHAX and chromosome region maintenance 1 and the box C/D snoRNP core protein fibrillarin concentrate in CBs from SMA cells, whereas the box H/ACA core proteins GAR1 and NAP57/dyskerin show reduced CB localization. Remarkably, the functional deficiency in SMA cells is associated with decreased localization of the snoRNP chaperone Nopp140 in CBs that correlates with disease severity. Indeed, RNA interference knockdown experiments in control fibroblasts demonstrate that SMN is required for accumulation of Nopp140 in CBs. Conversely, overexpression of SMN in SMA cells restores the CB localization of Nopp140, whereas SMN mutants found in SMA patients are defective in promoting the association of Nopp140 with CBs. Taken together, we demonstrate that only a subset of CB functions (as indicated by the association of representative factors) are impaired in SMA cells and, importantly, we identify the decrease of Nopp140 localization in CBs as a phenotypic marker for SMA.

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Figures

Figure 1.
Figure 1.
Subnuclear localization of CRM1, PHAX, C/D box snoRNP component fibrillarin, H/ACA box snoRNP proteins GAR1 and NAP57/dyskerin, snoRNP chaperone Nopp140 and coilin in SMA-derived fibroblast cells. Immortalized control and Type I SMA patient fibroblasts were costained for CRM1 (A), PHAX (B), fibrillarin (C), GAR 1 (D), NAP57/dyskerin (E) or Nopp140 (F) in green and the CB marker coilin in red. CRM1 and PHAX nuclear foci in both control and SMA cells were localized in CBs, as shown in yellow (merge). The arrow points to one of the CBs. It should be noted for both control and SMA cells that most of the immunolabelling for fibrillarin, GAR1, NAP57/dyskerin or Nopp140 concentrates in nucleoli (arrowhead) as expected. Fibrillarin nuclear foci (arrow) in both control and SMA cells were localized with coilin in CBs, as shown in yellow. GAR1, NAP57/dyskerin and Nopp140 nuclear foci (arrow) were found colocalized with coilin in control cells. In contrast, only a few Nopp140 foci (arrow) were detected in SMA cells and were not colocalized with coilin. The arrowhead and arrow point to one of the nucleoli and one of the CBs, respectively. Scale bars, 3 µm.
Figure 2.
Figure 2.
Analyses of the subnuclear localization of the snoRNP chaperone Nopp140 in CBs from control and SMA cells. The results in the histogram (A) and table (B) demonstrated the marked difference between control and Type I SMA-derived cells. Note the right-hand part of the table in (B) refers to the Nopp140-positive foci (44% in control and 10% in SMAI) as 100%.
Figure 3.
Figure 3.
Subnuclear localization of SMN and Nopp140 observed upon transient transfection of immortalized Type I SMA-derived fibroblast cells. The SMA cells were transiently transfected with eGFP-SMN (A). The arrowhead and arrow point to one of the nucleoli and one of the CBs, respectively. The distribution of SMN and Nopp140 foci were compared with untransfected cells (B). Scale bars, 3 µm.
Figure 4.
Figure 4.
SMN and Nopp140 RNA interference. The double immunolabelling experiments for SMN and Nopp140 were performed in immortalized control human fibroblasts 48 h after transfected with SMN or Nopp140 siRNA (A). The arrowhead and arrow point to one of the nucleoli and one of the CBs, respectively. The asterisk points to an unsilenced cell. The statistical analyses are presented in table (B). Western blot analyses of protein extracts from cells treated with siRNA to scrambled, SMN or Nopp140 revealed the specificity of knockdown (C). The α-tubulin incubation served as loading control. Scale bars, 3 µm.
Figure 5.
Figure 5.
Comparison of the localization of Nopp140 in CBs of cells transfected with wild-type SMN and SMA mutants. The SMN protein is depicted with the Lys-rich, Tudor, Pro-rich, oligomerization YG-box and ex7 encoded domains. SMN and SMA mutants were fused to the N-terminal end of eGFP (A, 36). The constructs indicated were transiently transfected into COS cells (B). Overexpression of eGFP-SMN (green) enhances the localization of Nopp140 to CBs (red), as observed in yellow (merge). The arrow points to one of the CBs. While the SMA mutants SMNE134K, Δex7 and 472Δ5 do not. The data are quantified in table (C). Scale bars, 3 µm.
Figure 6.
Figure 6.
Subnuclear localization of Nopp140 in primary fibroblasts derived from control and Type I, II and III SMA patients. Primary fibroblasts were immunostained for two CB markers SMN (A) or coilin (B) in green and Nopp140 in red. Colocalization in CBs (arrow) is shown in yellow (merge). The arrowhead and arrow point to one of the nucleoli and one of the CBs, respectively. The statistical analyses show a correlation between Nopp140-positive CBs and SMA disease severity and are presented in a histogram (C) and in Table 2. Scale bars, 3 µm.
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