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. 2013 Oct;9(10):e1003890.
doi: 10.1371/journal.pgen.1003890. Epub 2013 Oct 31.

Nuclear Fragile X Mental Retardation Protein is localized to Cajal bodies

Alain Y Dury  1 , Rachid El Fatimy, Sandra Tremblay, Timothy M Rose, Jocelyn Côté, Paul De Koninck, Edouard W Khandjian
Affiliations

Nuclear Fragile X Mental Retardation Protein is localized to Cajal bodies

Alain Y Dury et al. PLoS Genet. 2013 Oct.

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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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. FMRP is present in Cajal bodies.
(A) Double immunofluorescence staining of HeLa cells with mAb1C3 (green) and IgY#C10 (red). Note that IgYC10 reveals intranuclear dots that are not seen with mAb1C3. (B) IgYC10 is specific to FMRP in normal human fibroblasts and does not stain any structure in Fragile X fibroblasts derived from a Fragile X donor. Nuclei were stained with DAPI. (C) Colocalization of nuclear FMRP (red) with Coilin (green). (D) Colocalization of nuclear FMRP (red) with SMN (green).
Figure 2
Figure 2. 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.
Figure 3
Figure 3. Effects of Leptomycin B on nuclear FMRP localization.
Hela cells were maintained in normal conditions (A) or treated with 2 ng/ml LMB for 18 h (B), and then processed for immunofluorescence to localize FMRP (red) and Coilin (green). Nuclei were counterstained with DAPI.
Figure 4
Figure 4. Nuclear ISO6 and ISO12 FMRP are present in Cajal bodies.
(A) Structural comparisons between the longest isoform 1 (ISO1) and the nuclear isoforms lacking the NES and RGG domains. Note that all isoforms lacking the NES domain (exon 14) have C-termini different (highlighted in gray) from the main FMRP isoform, due to frame shifts. Note also that mAb1C3 detects all FMRP isoforms. (B) Localization of cytoplasmic and nuclear FMRP isoforms tagged with GFP, after transfection of HeLa cells with the corresponding expression vectors. Note the presence of ISO6 and ISO12 in Cajal bodies, while ISO1 is exclusively localized in the cytoplasm. (C) GFP-ISO6 FMRP colocalizes with Coilin in Cajal bodies (white arrow heads).
Figure 5
Figure 5. 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 ; Pan troglodytes ref|XP_003317790.1|; Mus Musculus (this study). (B) C-terminal amino acid sequences of hISO6 and a mouse ISO6 variant 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.
Figure 6
Figure 6. 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.
Figure 7
Figure 7. 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).
Figure 8
Figure 8. 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.
Figure 9
Figure 9. ISO6-I304N FMRP is defective in its Cajal body association.
(A) GFP-ISO6-I304N displays reduced association with Cajal bodies. Note that not all coilin positive structures contain ISO6-I304N and that the majority of the ISO6-I304N remains nucleoplasmic. Arrowhead point to mini-ISO6-I304N positive Cajal bodies. (B) HeLa cells transiently transfected with the indicated GFP-fusion construct were used for FRAP experiments (as described in Material and Methods). (C) GFP-ISO6-I304N displays a faster turnover rate relative to wild type FMRP-ISO6.
Figure 10
Figure 10. Proposed model for the fate of the nuclear ISO6 and full length ISO1 FMRP.
(A) Schematic representation of ISO1 and ISO6 FMRP and mapping of known protein partners. Note the position of the Cajal Body Localization Signal (CBLS). (B) Alternative splicing of the primary transcripts generates either ISO6 FMRP lacking the CRD domain, or ISO1 FMRP containing both NLS and CRD domains. ISO6 is driven to Cajal bodies by transporter proteins, while ISO1 interacts with protein partners that lock the NLS and CRD domains and is localized to the perinuclear area to join the nascent mRNPs complexes emerging from the nuclear pores. In the cytoplasm the ISO1 FMRP-mRNPs particles associate with the translation machinery or are transported in RNA-granules to micro-domains away from the soma.
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