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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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FMRP is present in the isolated nuclear fraction but not in nuclei.(A) Total, cytoplasmic, and nuclear cytoplasmic fractions from HeLa cells were loaded in equal ratios as well as one overloaded nuclear fraction and analyzed by immunoblotting with mAb1C3 to determine the distribution of FMRP. Nuc+ refers to concentrated (20 µg) nuclear protein. (B) Double immunofluorescent localization of FMRP with IgYC10 (red) and Coilin (green) after gentle lysis of the cells in situ. Nuclei were counterstained with DAPI. (C) Double immunofluorescent staining of FMRP with IgYC10 (red) and cold-resistant microtubule network revealed with an anti-tubulin antibody (green). Nuclei were counterstained with DAPI. Due to the three dimensional distribution of microtubules, images were taken by conventional epifluorescent microscopy to reveal the microtubule framework.
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pgen-1003890-g002: FMRP is present in the isolated nuclear fraction but not in nuclei.(A) Total, cytoplasmic, and nuclear cytoplasmic fractions from HeLa cells were loaded in equal ratios as well as one overloaded nuclear fraction and analyzed by immunoblotting with mAb1C3 to determine the distribution of FMRP. Nuc+ refers to concentrated (20 µg) nuclear protein. (B) Double immunofluorescent localization of FMRP with IgYC10 (red) and Coilin (green) after gentle lysis of the cells in situ. Nuclei were counterstained with DAPI. (C) Double immunofluorescent staining of FMRP with IgYC10 (red) and cold-resistant microtubule network revealed with an anti-tubulin antibody (green). Nuclei were counterstained with DAPI. Due to the three dimensional distribution of microtubules, images were taken by conventional epifluorescent microscopy to reveal the microtubule framework.

Mentions: The observation that FMRP localizes in Cajal bodies raised the question of whether FMRP is only transiently present in the nucleus as part of a shuttling process, or if a specific FMRP sub-population is targeted to the Cajal bodies to remain there. Using standard cell fractionation analyses, it has been estimated that 5 to 10% of total FMRP is recovered within the nuclear fraction (Figure 2A and [15]). However, when we increased the concentration of the nuclear sample (∼20 µg as for the total sample), additional bands of lower molecular weights could be detected (Figure 2A). Either these bands corresponded to new FMRP species, or to degradation products. To our knowledge it has not been proven yet whether this so-called nuclear FMRP is present inside the nucleus or is associated with the nuclear enriched fraction obtained after cell lysis as pointed out by Sittler et al[17]. To investigate this question, HeLa cells grown on coverslips were lysed in situ in the presence of a buffer containing the non-ionic detergent NP40 to remove most of the cytoplasm, while a cold-resistant cytoskeletal framework containing the cell nucleus [24]–[26] remained attached to the coverslip. After such a treatment, we observed that the FMRP cytoplasmic staining detected with IgYC10 was greatly reduced and was mainly present as perinuclear granular structures outside of the nucleus (Figure 2B) embedded in the cytoskeleton framework, as highlighted using an anti-tubulin antibody (Figure 2C). The same cytoplasmic distribution was also observed using mAb1C3 (Figure S1). On the other hand, FMRP-containing nuclear foci were still detectable following this treatment, strongly arguing that these are indeed nuclear structures.


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)

FMRP is present in the isolated nuclear fraction but not in nuclei.(A) Total, cytoplasmic, and nuclear cytoplasmic fractions from HeLa cells were loaded in equal ratios as well as one overloaded nuclear fraction and analyzed by immunoblotting with mAb1C3 to determine the distribution of FMRP. Nuc+ refers to concentrated (20 µg) nuclear protein. (B) Double immunofluorescent localization of FMRP with IgYC10 (red) and Coilin (green) after gentle lysis of the cells in situ. Nuclei were counterstained with DAPI. (C) Double immunofluorescent staining of FMRP with IgYC10 (red) and cold-resistant microtubule network revealed with an anti-tubulin antibody (green). Nuclei were counterstained with DAPI. Due to the three dimensional distribution of microtubules, images were taken by conventional epifluorescent microscopy to reveal the microtubule framework.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003890-g002: FMRP is present in the isolated nuclear fraction but not in nuclei.(A) Total, cytoplasmic, and nuclear cytoplasmic fractions from HeLa cells were loaded in equal ratios as well as one overloaded nuclear fraction and analyzed by immunoblotting with mAb1C3 to determine the distribution of FMRP. Nuc+ refers to concentrated (20 µg) nuclear protein. (B) Double immunofluorescent localization of FMRP with IgYC10 (red) and Coilin (green) after gentle lysis of the cells in situ. Nuclei were counterstained with DAPI. (C) Double immunofluorescent staining of FMRP with IgYC10 (red) and cold-resistant microtubule network revealed with an anti-tubulin antibody (green). Nuclei were counterstained with DAPI. Due to the three dimensional distribution of microtubules, images were taken by conventional epifluorescent microscopy to reveal the microtubule framework.
Mentions: The observation that FMRP localizes in Cajal bodies raised the question of whether FMRP is only transiently present in the nucleus as part of a shuttling process, or if a specific FMRP sub-population is targeted to the Cajal bodies to remain there. Using standard cell fractionation analyses, it has been estimated that 5 to 10% of total FMRP is recovered within the nuclear fraction (Figure 2A and [15]). However, when we increased the concentration of the nuclear sample (∼20 µg as for the total sample), additional bands of lower molecular weights could be detected (Figure 2A). Either these bands corresponded to new FMRP species, or to degradation products. To our knowledge it has not been proven yet whether this so-called nuclear FMRP is present inside the nucleus or is associated with the nuclear enriched fraction obtained after cell lysis as pointed out by Sittler et al[17]. To investigate this question, HeLa cells grown on coverslips were lysed in situ in the presence of a buffer containing the non-ionic detergent NP40 to remove most of the cytoplasm, while a cold-resistant cytoskeletal framework containing the cell nucleus [24]–[26] remained attached to the coverslip. After such a treatment, we observed that the FMRP cytoplasmic staining detected with IgYC10 was greatly reduced and was mainly present as perinuclear granular structures outside of the nucleus (Figure 2B) embedded in the cytoskeleton framework, as highlighted using an anti-tubulin antibody (Figure 2C). The same cytoplasmic distribution was also observed using mAb1C3 (Figure S1). On the other hand, FMRP-containing nuclear foci were still detectable following this treatment, strongly arguing that these are indeed nuclear structures.

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