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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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The Cajal body localization signal of human ISO6 is localized to a 17aa C-terminal domain.(A) Evolutionary conserved C-termini of FMRP ISO6. ClustalW multiple sequence alignment of predicted FMRP ISO6 isoforms from different organisms compared to the experimentally determined human ISO6 sequence. Exon positions and numbering are indicated (see Figure S4). GenBank accession numbers : Sus scrofa ref/XP_003360519.1/; Felis catus ref/XP_004000999.1/; Ovis aries ref/XP_004022340.1/; Saimiri boliviensis boliviensis ref/XP_003939137.1/; Canis lupus familiaris ref/XP_003435591.1/; Nomascus leucogenys ref/XP_003271865.1/; Homo sapiens ref/NP_001172004.1/ and [17]; Pan troglodytes ref/XP_003317790.1/; Mus Musculus (this study). (B) C-terminal amino acid sequences of hISO6 [17] and a mouse ISO6 variant [35] determined from cloned cDNAs. (C) Schematic representation of GFP hybrids of full-length human ISO6 and the shorter murine ISO6 variant (mIso6) and the GFP-human ISO6 with the 17aa deletion and their localization (D) in human HeLa and murine MN-1 cells after transient transfection.
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pgen-1003890-g005: The Cajal body localization signal of human ISO6 is localized to a 17aa C-terminal domain.(A) Evolutionary conserved C-termini of FMRP ISO6. ClustalW multiple sequence alignment of predicted FMRP ISO6 isoforms from different organisms compared to the experimentally determined human ISO6 sequence. Exon positions and numbering are indicated (see Figure S4). GenBank accession numbers : Sus scrofa ref/XP_003360519.1/; Felis catus ref/XP_004000999.1/; Ovis aries ref/XP_004022340.1/; Saimiri boliviensis boliviensis ref/XP_003939137.1/; Canis lupus familiaris ref/XP_003435591.1/; Nomascus leucogenys ref/XP_003271865.1/; Homo sapiens ref/NP_001172004.1/ and [17]; Pan troglodytes ref/XP_003317790.1/; Mus Musculus (this study). (B) C-terminal amino acid sequences of hISO6 [17] and a mouse ISO6 variant [35] determined from cloned cDNAs. (C) Schematic representation of GFP hybrids of full-length human ISO6 and the shorter murine ISO6 variant (mIso6) and the GFP-human ISO6 with the 17aa deletion and their localization (D) in human HeLa and murine MN-1 cells after transient transfection.

Mentions: Previously, sequence analysis of a cDNA encoding human ISO6 [17] revealed that this isoform was generated by alternative splicing of FMR1 pre-mRNA in which exon 13 was spliced directly to exon 15 at a distal splice acceptor site (Figure S4A), eliminating exon 14 and the sequences encoded in exon 15 from the region between the proximal and distal splice acceptor sites (labeled exon 15a), which are present in ISO1 (reading frame RF3; Figure S4A). Splicing of exon 13 to 15 in ISO6 results in an amino acid sequence derived from reading frame 1 in exon 15b from the distal splice acceptor site (Figure 5A and Figure S4A). This alternate reading frame continues through exon 16 which is spliced to exon 17 (Figure S4B). In human ISO6 cDNA, exon 16 is spliced to exon 17 using a proximal splice acceptor site generating a transcript that encodes an amino sequence in reading frame 2 (RF2; Figure S4C). The alternative splicing detected in the human ISO6 cDNA determined by Sittler et al. [17] is predicted to occur in a variety of other species, as shown in Figure 5A. ISO12 is similar to ISO6 except that it lacks exon 12, which results in shortening of a loop between the β2 and β′ strands within the KH2 domain [36].


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)

The Cajal body localization signal of human ISO6 is localized to a 17aa C-terminal domain.(A) Evolutionary conserved C-termini of FMRP ISO6. ClustalW multiple sequence alignment of predicted FMRP ISO6 isoforms from different organisms compared to the experimentally determined human ISO6 sequence. Exon positions and numbering are indicated (see Figure S4). GenBank accession numbers : Sus scrofa ref/XP_003360519.1/; Felis catus ref/XP_004000999.1/; Ovis aries ref/XP_004022340.1/; Saimiri boliviensis boliviensis ref/XP_003939137.1/; Canis lupus familiaris ref/XP_003435591.1/; Nomascus leucogenys ref/XP_003271865.1/; Homo sapiens ref/NP_001172004.1/ and [17]; Pan troglodytes ref/XP_003317790.1/; Mus Musculus (this study). (B) C-terminal amino acid sequences of hISO6 [17] and a mouse ISO6 variant [35] determined from cloned cDNAs. (C) Schematic representation of GFP hybrids of full-length human ISO6 and the shorter murine ISO6 variant (mIso6) and the GFP-human ISO6 with the 17aa deletion and their localization (D) in human HeLa and murine MN-1 cells after transient transfection.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814324&req=5

pgen-1003890-g005: The Cajal body localization signal of human ISO6 is localized to a 17aa C-terminal domain.(A) Evolutionary conserved C-termini of FMRP ISO6. ClustalW multiple sequence alignment of predicted FMRP ISO6 isoforms from different organisms compared to the experimentally determined human ISO6 sequence. Exon positions and numbering are indicated (see Figure S4). GenBank accession numbers : Sus scrofa ref/XP_003360519.1/; Felis catus ref/XP_004000999.1/; Ovis aries ref/XP_004022340.1/; Saimiri boliviensis boliviensis ref/XP_003939137.1/; Canis lupus familiaris ref/XP_003435591.1/; Nomascus leucogenys ref/XP_003271865.1/; Homo sapiens ref/NP_001172004.1/ and [17]; Pan troglodytes ref/XP_003317790.1/; Mus Musculus (this study). (B) C-terminal amino acid sequences of hISO6 [17] and a mouse ISO6 variant [35] determined from cloned cDNAs. (C) Schematic representation of GFP hybrids of full-length human ISO6 and the shorter murine ISO6 variant (mIso6) and the GFP-human ISO6 with the 17aa deletion and their localization (D) in human HeLa and murine MN-1 cells after transient transfection.
Mentions: Previously, sequence analysis of a cDNA encoding human ISO6 [17] revealed that this isoform was generated by alternative splicing of FMR1 pre-mRNA in which exon 13 was spliced directly to exon 15 at a distal splice acceptor site (Figure S4A), eliminating exon 14 and the sequences encoded in exon 15 from the region between the proximal and distal splice acceptor sites (labeled exon 15a), which are present in ISO1 (reading frame RF3; Figure S4A). Splicing of exon 13 to 15 in ISO6 results in an amino acid sequence derived from reading frame 1 in exon 15b from the distal splice acceptor site (Figure 5A and Figure S4A). This alternate reading frame continues through exon 16 which is spliced to exon 17 (Figure S4B). In human ISO6 cDNA, exon 16 is spliced to exon 17 using a proximal splice acceptor site generating a transcript that encodes an amino sequence in reading frame 2 (RF2; Figure S4C). The alternative splicing detected in the human ISO6 cDNA determined by Sittler et al. [17] is predicted to occur in a variety of other species, as shown in Figure 5A. ISO12 is similar to ISO6 except that it lacks exon 12, which results in shortening of a loop between the β2 and β′ strands within the KH2 domain [36].

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