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Nuclear Fragile X Mental Retardation Protein is localized to Cajal bodies.

Dury AY, El Fatimy R, Tremblay S, Rose TM, Côté J, De Koninck P, Khandjian EW - PLoS Genet. (2013)

Bottom Line: However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process.Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7) do not localize to the nucleus.Supporting this hypothesis, a missense mutation (I304N), known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies.

View Article: PubMed Central - PubMed

Affiliation: Centre de recherche, Institut en santé mentale de Québec, Québec, Québec, Canada ; Département de psychiatrie et des neurosciences, Faculté de médecine, Université Laval, Québec, Québec, Canada.

ABSTRACT
Fragile X syndrome is caused by loss of function of a single gene encoding the Fragile X Mental Retardation Protein (FMRP). This RNA-binding protein, widely expressed in mammalian tissues, is particularly abundant in neurons and is a component of messenger ribonucleoprotein (mRNP) complexes present within the translational apparatus. The absence of FMRP in neurons is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. A prevalent model posits that FMRP is a nucleocytoplasmic shuttling protein that transports its mRNA targets from the nucleus to the translation machinery. However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process. Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7) do not localize to the nucleus. Instead, specific FMRP isoforms 6 and 12 (ISO6 and 12), containing a novel C-terminal domain, were the only isoforms that localized to the nuclei in cultured human cells. These isoforms localized to specific p80-coilin and SMN positive structures that were identified as Cajal bodies. The Cajal body localization signal was confined to a 17 amino acid stretch in the C-terminus of human ISO6 and is lacking in a mouse Iso6 variant. As FMRP is an RNA-binding protein, its presence in Cajal bodies suggests additional functions in nuclear post-transcriptional RNA metabolism. Supporting this hypothesis, a missense mutation (I304N), known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies. These findings open unexplored avenues in search for new insights into the pathophysiology of Fragile X Syndrome.

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Effects of Leptomycin B on cytoplasmic and nuclear GFP-FMRP localizations.Control and transfected HeLa cells with vectors coding for GFP-ISO7 (A) and GFP-ISO6 (B) were maintained in normal conditions or treated with 2 ng/ml LMB for 20 h, and then processed for immunofluorescence to localize FMRP (green) and Coilin (red). Nuclei were stained with DAPI.
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pgen-1003890-g006: Effects of Leptomycin B on cytoplasmic and nuclear GFP-FMRP localizations.Control and transfected HeLa cells with vectors coding for GFP-ISO7 (A) and GFP-ISO6 (B) were maintained in normal conditions or treated with 2 ng/ml LMB for 20 h, and then processed for immunofluorescence to localize FMRP (green) and Coilin (red). Nuclei were stained with DAPI.

Mentions: Having shown that endogenous cytoplasmic FMRP does not appear to traffic to the nucleus after treatment with LMB (see above), we confirmed these results using transfection assays. HeLa cells were transfected with vectors encoding GFP-ISO7 and GFP-ISO6. Four hours after transfection, LMB was added to the culture medium at a concentration of 2 ng/ml. After 20 hours of treatment with the drug, cells were processed for immunofluorescence analyses. Control cells with no LMB treatment showed strong GFP-ISO7 cytoplasmic fluorescence and as expected, the presence of FMRP in stress granules due to over-accumulation [29]. The presence of Cajal bodies in the nucleus was assessed using anti-coilin staining, and no difference could be observed between transfected and untransfected cells (Figure 6A). In contrast, LMB treatment induced Cajal bodies to become dispersed, as numerous smaller coilin positive foci were redistributed in the nucleoplasm in both transfected and untransfected cells (Figure 6A). On the other hand, no transfected GFP-ISO7 could be detected in the nucleus after LMB treatment, confirming our previous results with endogenous FMRP (see Figure 3). We next tested the effects of LMB on the nuclear distribution of ISO6. In untreated cells, GFP-ISO6 was associated mainly with Cajal bodies similarly to the endogenous nuclear FMRP detected with the IgYC10 antibody (see Figure 1). After treatment with LMB, GFP-ISO6 was no longer concentrated in Cajal bodies, as it was evenly distributed throughout the nucleoplasm. Also, coilin was redistributed in the nucleoplasm, as smaller coilin positive foci were observed (Figure 6B).


Nuclear Fragile X Mental Retardation Protein is localized to Cajal bodies.

Dury AY, El Fatimy R, Tremblay S, Rose TM, Côté J, De Koninck P, Khandjian EW - PLoS Genet. (2013)

Effects of Leptomycin B on cytoplasmic and nuclear GFP-FMRP localizations.Control and transfected HeLa cells with vectors coding for GFP-ISO7 (A) and GFP-ISO6 (B) were maintained in normal conditions or treated with 2 ng/ml LMB for 20 h, and then processed for immunofluorescence to localize FMRP (green) and Coilin (red). Nuclei were stained with DAPI.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814324&req=5

pgen-1003890-g006: Effects of Leptomycin B on cytoplasmic and nuclear GFP-FMRP localizations.Control and transfected HeLa cells with vectors coding for GFP-ISO7 (A) and GFP-ISO6 (B) were maintained in normal conditions or treated with 2 ng/ml LMB for 20 h, and then processed for immunofluorescence to localize FMRP (green) and Coilin (red). Nuclei were stained with DAPI.
Mentions: Having shown that endogenous cytoplasmic FMRP does not appear to traffic to the nucleus after treatment with LMB (see above), we confirmed these results using transfection assays. HeLa cells were transfected with vectors encoding GFP-ISO7 and GFP-ISO6. Four hours after transfection, LMB was added to the culture medium at a concentration of 2 ng/ml. After 20 hours of treatment with the drug, cells were processed for immunofluorescence analyses. Control cells with no LMB treatment showed strong GFP-ISO7 cytoplasmic fluorescence and as expected, the presence of FMRP in stress granules due to over-accumulation [29]. The presence of Cajal bodies in the nucleus was assessed using anti-coilin staining, and no difference could be observed between transfected and untransfected cells (Figure 6A). In contrast, LMB treatment induced Cajal bodies to become dispersed, as numerous smaller coilin positive foci were redistributed in the nucleoplasm in both transfected and untransfected cells (Figure 6A). On the other hand, no transfected GFP-ISO7 could be detected in the nucleus after LMB treatment, confirming our previous results with endogenous FMRP (see Figure 3). We next tested the effects of LMB on the nuclear distribution of ISO6. In untreated cells, GFP-ISO6 was associated mainly with Cajal bodies similarly to the endogenous nuclear FMRP detected with the IgYC10 antibody (see Figure 1). After treatment with LMB, GFP-ISO6 was no longer concentrated in Cajal bodies, as it was evenly distributed throughout the nucleoplasm. Also, coilin was redistributed in the nucleoplasm, as smaller coilin positive foci were observed (Figure 6B).

Bottom Line: However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process.Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7) do not localize to the nucleus.Supporting this hypothesis, a missense mutation (I304N), known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies.

View Article: PubMed Central - PubMed

Affiliation: Centre de recherche, Institut en santé mentale de Québec, Québec, Québec, Canada ; Département de psychiatrie et des neurosciences, Faculté de médecine, Université Laval, Québec, Québec, Canada.

ABSTRACT
Fragile X syndrome is caused by loss of function of a single gene encoding the Fragile X Mental Retardation Protein (FMRP). This RNA-binding protein, widely expressed in mammalian tissues, is particularly abundant in neurons and is a component of messenger ribonucleoprotein (mRNP) complexes present within the translational apparatus. The absence of FMRP in neurons is believed to cause translation dysregulation and defects in mRNA transport essential for local protein synthesis and for synaptic development and maturation. A prevalent model posits that FMRP is a nucleocytoplasmic shuttling protein that transports its mRNA targets from the nucleus to the translation machinery. However, it is not known which of the multiple FMRP isoforms, resulting from the numerous alternatively spliced FMR1 transcripts variants, would be involved in such a process. Using a new generation of anti-FMRP antibodies and recombinant expression, we show here that the most commonly expressed human FMRP isoforms (ISO1 and 7) do not localize to the nucleus. Instead, specific FMRP isoforms 6 and 12 (ISO6 and 12), containing a novel C-terminal domain, were the only isoforms that localized to the nuclei in cultured human cells. These isoforms localized to specific p80-coilin and SMN positive structures that were identified as Cajal bodies. The Cajal body localization signal was confined to a 17 amino acid stretch in the C-terminus of human ISO6 and is lacking in a mouse Iso6 variant. As FMRP is an RNA-binding protein, its presence in Cajal bodies suggests additional functions in nuclear post-transcriptional RNA metabolism. Supporting this hypothesis, a missense mutation (I304N), known to alter the KH2-mediated RNA binding properties of FMRP, abolishes the localization of human FMRP ISO6 to Cajal bodies. These findings open unexplored avenues in search for new insights into the pathophysiology of Fragile X Syndrome.

Show MeSH
Related in: MedlinePlus