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

Dury AY, El Fatimy R, Tremblay S, Rose TM, CÎté 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 binds to RNA homopolymers.Extracts from STEK Fmr1−/− KO cells expressing ISO6 (A) and ISO7 (B) were mixed with agarose beads carrying polyA, polyU, polyC and polyG homopolymers. Captured proteins in the presence of 150 and 300 mM NaCl were eluted with SDS-sample buffer and analyzed by immunoblotting using mAb1C3. (C) Cleaved ISO6 in Cajal bodies also binds preferentially to polyG and to a lesser extend to polyU as is the case for ISO7 and ISO1 (not shown) FMRP.
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pgen-1003890-g008: ISO6 binds to RNA homopolymers.Extracts from STEK Fmr1−/− KO cells expressing ISO6 (A) and ISO7 (B) were mixed with agarose beads carrying polyA, polyU, polyC and polyG homopolymers. Captured proteins in the presence of 150 and 300 mM NaCl were eluted with SDS-sample buffer and analyzed by immunoblotting using mAb1C3. (C) Cleaved ISO6 in Cajal bodies also binds preferentially to polyG and to a lesser extend to polyU as is the case for ISO7 and ISO1 (not shown) FMRP.

Mentions: In the absence of any information about putative RNAs that would bind ISO6, we performed RNA binding assays using homopolymer RNAs conjugated to agarose beads [45] to determine the ability of ISO6 to bind RNA compared to its full-length ISO1 counterpart. Despite the fact that it lacks the C-terminal RGG domain present in ISO1, we observed that ISO6 was preferentially retained on polyG and to a lesser extent to polyU, but not to polyA or polyC (Figure 8B), a pattern of binding to RNA homopolymers similar to that observed for FMRP ISO1 [5]. Since our studies suggested that ISO6 is processed in Cajal bodies to yield a 44 kDa protein lacking the C-terminus, but still retaining the KH1 and KH2 domains, we examined the RNA binding of the cleaved ISO6 from extracted Cajal bodies. We observed the same binding patterns seen with full length ISO6. The binding activity to polyG and polyU was still stable at 300 mM NaCl. These results strongly suggest that the affinity of ISO6 to polyG is not due to the RGG domain, but rather to the intrinsic properties of the KH1 and KH2 domains, that are shared by the different FMRP isoforms. Our results suggest that ISO6 might also play a role in interacting with RNA in Cajal bodies.


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

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

ISO6 binds to RNA homopolymers.Extracts from STEK Fmr1−/− KO cells expressing ISO6 (A) and ISO7 (B) were mixed with agarose beads carrying polyA, polyU, polyC and polyG homopolymers. Captured proteins in the presence of 150 and 300 mM NaCl were eluted with SDS-sample buffer and analyzed by immunoblotting using mAb1C3. (C) Cleaved ISO6 in Cajal bodies also binds preferentially to polyG and to a lesser extend to polyU as is the case for ISO7 and ISO1 (not shown) FMRP.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003890-g008: ISO6 binds to RNA homopolymers.Extracts from STEK Fmr1−/− KO cells expressing ISO6 (A) and ISO7 (B) were mixed with agarose beads carrying polyA, polyU, polyC and polyG homopolymers. Captured proteins in the presence of 150 and 300 mM NaCl were eluted with SDS-sample buffer and analyzed by immunoblotting using mAb1C3. (C) Cleaved ISO6 in Cajal bodies also binds preferentially to polyG and to a lesser extend to polyU as is the case for ISO7 and ISO1 (not shown) FMRP.
Mentions: In the absence of any information about putative RNAs that would bind ISO6, we performed RNA binding assays using homopolymer RNAs conjugated to agarose beads [45] to determine the ability of ISO6 to bind RNA compared to its full-length ISO1 counterpart. Despite the fact that it lacks the C-terminal RGG domain present in ISO1, we observed that ISO6 was preferentially retained on polyG and to a lesser extent to polyU, but not to polyA or polyC (Figure 8B), a pattern of binding to RNA homopolymers similar to that observed for FMRP ISO1 [5]. Since our studies suggested that ISO6 is processed in Cajal bodies to yield a 44 kDa protein lacking the C-terminus, but still retaining the KH1 and KH2 domains, we examined the RNA binding of the cleaved ISO6 from extracted Cajal bodies. We observed the same binding patterns seen with full length ISO6. The binding activity to polyG and polyU was still stable at 300 mM NaCl. These results strongly suggest that the affinity of ISO6 to polyG is not due to the RGG domain, but rather to the intrinsic properties of the KH1 and KH2 domains, that are shared by the different FMRP isoforms. Our results suggest that ISO6 might also play a role in interacting with RNA in Cajal bodies.

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