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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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ISO6 FMRP is cleaved by calpain in isolated Cajal bodies.(A) The detected FMRP associated with the Cajal bodies has an apparent molecular weight lower than expected for ISO6 FMRP. Immunoblot analyses of FMRP present in Cajal bodies using different antibodies to FMRP. (B) ISO6 FMRP is a substrate for calpain1. Assays were carried out either with immunoprecipitated ISO6 FMRP or with total cell lysate in the presence of 0.05 U of Calpain 1 for 10 and 20 min at room temperature. The reaction was inhibited in the presence of ALLN (+ Inhib). C : control reaction without the enzyme. (C) Comparison of cleavage products and intermediates between ISO6 and ISO7 FMRP. FMRP species were revealed with four different antibodies in (A) and with mAb1C3 in (B,C).
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pgen-1003890-g007: ISO6 FMRP is cleaved by calpain in isolated Cajal bodies.(A) The detected FMRP associated with the Cajal bodies has an apparent molecular weight lower than expected for ISO6 FMRP. Immunoblot analyses of FMRP present in Cajal bodies using different antibodies to FMRP. (B) ISO6 FMRP is a substrate for calpain1. Assays were carried out either with immunoprecipitated ISO6 FMRP or with total cell lysate in the presence of 0.05 U of Calpain 1 for 10 and 20 min at room temperature. The reaction was inhibited in the presence of ALLN (+ Inhib). C : control reaction without the enzyme. (C) Comparison of cleavage products and intermediates between ISO6 and ISO7 FMRP. FMRP species were revealed with four different antibodies in (A) and with mAb1C3 in (B,C).

Mentions: To demonstrate biochemically the presence of ISO6 and ISO12 FMRP in Cajal bodies, we isolated and purified these structures according to the procedure described by the Lamond's laboratory [38], [39]. Immunoblot analyses of Cajal body proteins using the FMRP mAb1C3 revealed a band at approximately 44 kDa (Figure 7A). The same band was observed using mAb2F5 directed against an epitope laying between amino acids 1 and 204 of FMRP [40], as well as with IgYC10. All three of these antibodies react with ISO1, 6, 7 and 12. In contrast, a rabbit polyclonal antibody directed against the FMRP peptide RTGKDRNQKKEKPD (amino acids 557 to 619) present at the C-terminus of full-length FMRP (ISO1) did not react with the Cajal extracts. Since this peptide sequence is present in ISO1 and 7, but not in ISO6 and 12, due to the frameshift induced by alternative splicing of exon 14, these results indicate that only ISO6/12 forms of FMRP associate with Cajal bodies. The unexpected observation that reactive FMRP in isolated Cajal bodies migrates at 44 kDa strongly suggests that ISO6/12 nuclear proteins are processed. Such a processing has been recently described for two well-known Cajal bodies markers, namely SMN and Coilin, which have been shown to be targets of calpain [41]–[43]. Contrary to proteases that fully degrade a substrate protein, calpains are calcium-dependent cysteine proteases that act by limited specific cleavages. We therefore examined whether the 44 kDa FMRP reactive protein could correspond to ISO6/12 that had undergone limited cleavage of the full length proteins, which have apparent molecular weights of 62 and 60 kDa respectively (see Figure S3). Bioinformatic searches [44] predict that the highest scoring calpain cleavage site is situated at amino acid 369, yielding an FMRP form with a theoretical molecular weight of 42 kDa, a value close to the observed apparent molecular weight of 44 kDa obtained in SDS-PAGE.


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)

ISO6 FMRP is cleaved by calpain in isolated Cajal bodies.(A) The detected FMRP associated with the Cajal bodies has an apparent molecular weight lower than expected for ISO6 FMRP. Immunoblot analyses of FMRP present in Cajal bodies using different antibodies to FMRP. (B) ISO6 FMRP is a substrate for calpain1. Assays were carried out either with immunoprecipitated ISO6 FMRP or with total cell lysate in the presence of 0.05 U of Calpain 1 for 10 and 20 min at room temperature. The reaction was inhibited in the presence of ALLN (+ Inhib). C : control reaction without the enzyme. (C) Comparison of cleavage products and intermediates between ISO6 and ISO7 FMRP. FMRP species were revealed with four different antibodies in (A) and with mAb1C3 in (B,C).
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pgen-1003890-g007: ISO6 FMRP is cleaved by calpain in isolated Cajal bodies.(A) The detected FMRP associated with the Cajal bodies has an apparent molecular weight lower than expected for ISO6 FMRP. Immunoblot analyses of FMRP present in Cajal bodies using different antibodies to FMRP. (B) ISO6 FMRP is a substrate for calpain1. Assays were carried out either with immunoprecipitated ISO6 FMRP or with total cell lysate in the presence of 0.05 U of Calpain 1 for 10 and 20 min at room temperature. The reaction was inhibited in the presence of ALLN (+ Inhib). C : control reaction without the enzyme. (C) Comparison of cleavage products and intermediates between ISO6 and ISO7 FMRP. FMRP species were revealed with four different antibodies in (A) and with mAb1C3 in (B,C).
Mentions: To demonstrate biochemically the presence of ISO6 and ISO12 FMRP in Cajal bodies, we isolated and purified these structures according to the procedure described by the Lamond's laboratory [38], [39]. Immunoblot analyses of Cajal body proteins using the FMRP mAb1C3 revealed a band at approximately 44 kDa (Figure 7A). The same band was observed using mAb2F5 directed against an epitope laying between amino acids 1 and 204 of FMRP [40], as well as with IgYC10. All three of these antibodies react with ISO1, 6, 7 and 12. In contrast, a rabbit polyclonal antibody directed against the FMRP peptide RTGKDRNQKKEKPD (amino acids 557 to 619) present at the C-terminus of full-length FMRP (ISO1) did not react with the Cajal extracts. Since this peptide sequence is present in ISO1 and 7, but not in ISO6 and 12, due to the frameshift induced by alternative splicing of exon 14, these results indicate that only ISO6/12 forms of FMRP associate with Cajal bodies. The unexpected observation that reactive FMRP in isolated Cajal bodies migrates at 44 kDa strongly suggests that ISO6/12 nuclear proteins are processed. Such a processing has been recently described for two well-known Cajal bodies markers, namely SMN and Coilin, which have been shown to be targets of calpain [41]–[43]. Contrary to proteases that fully degrade a substrate protein, calpains are calcium-dependent cysteine proteases that act by limited specific cleavages. We therefore examined whether the 44 kDa FMRP reactive protein could correspond to ISO6/12 that had undergone limited cleavage of the full length proteins, which have apparent molecular weights of 62 and 60 kDa respectively (see Figure S3). Bioinformatic searches [44] predict that the highest scoring calpain cleavage site is situated at amino acid 369, yielding an FMRP form with a theoretical molecular weight of 42 kDa, a value close to the observed apparent molecular weight of 44 kDa obtained in SDS-PAGE.

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