Fragile Mental Retardation Protein Interacts with the RNA-Binding Protein Caprin1 in Neuronal RiboNucleoProtein Complexes

Fragile X syndrome is caused by the absence of the Fragile X Mental Retardation Protein (FMRP), an RNA-binding protein. FMRP is associated with messenger RiboNucleoParticles (mRNPs) present in polyribosomes and its absence in neurons leads to alteration in synaptic plasticity as a result of translation regulation defects. The molecular mechanisms by which FMRP plays a role in translation regulation remain elusive. Using immunoprecipitation approaches with monoclonal Ab7G1-1 and a new generation of chicken antibodies, we identified Caprin1 as a novel FMRP-cellular partner. In vivo and in vitro evidence show that Caprin1 interacts with FMRP at the level of the translation machinery as well as in trafficking neuronal granules. As an RNA-binding protein, Caprin1 has in common with FMRP at least two RNA targets that have been identified as CaMKIIα and Map1b mRNAs. In view of the new concept that FMRP species bind to RNA regardless of known structural motifs, we propose that protein interactors might modulate FMRP functions

Nuclear Fragile X Mental Retardation Protein Is localized to Cajal Bodies

<div><p>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 <i>FMR1</i> 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.</p></div

Oligonucleotides primers used in RT-PCR analyses.

<p>Oligonucleotides primers used in RT-PCR analyses.</p

mRNAs associated with Caprin1 and FMRP. A

<p>) mRNAs co-immunoprecipitated by mAb7G1-1 and IgY#C10 from WT and KO brain extracts were analyzed by RT-PCR and visualized by agarose gels (left panels) and by Light Cycler RT-PCR (right panels). Bars in red refer to mRNA targets to Caprin1, in blue common to Caprin1 and FMRP, in green to FMRP and in black to non-targets mRNAs. Dark colors refer to WT and pale to KO, respectively. N = 5, P≤0.001 of a pool of 10 adult brains. Vertical black dot lines represent thresholds corresponding to background. <b>B</b>) RT-PCR analyses of selected mRNAs co-immunoprecipitated with Caprin1.</p

Proposed model for the fate of the nuclear ISO6 and full length ISO1 FMRP.

<p>(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.</p

The Cajal body localization signal of human ISO6 is localized to a 17aa C-terminal domain.

<p>(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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003890#pgen.1003890.s004" target="_blank">Figure S4</a>). 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003890#pgen.1003890-Sittler1" target="_blank">[17]</a>; Pan troglodytes ref|XP_003317790.1|; Mus Musculus (this study). (B) C-terminal amino acid sequences of hISO6 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003890#pgen.1003890-Sittler1" target="_blank">[17]</a> and a mouse ISO6 variant <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003890#pgen.1003890-Brackett1" target="_blank">[35]</a> 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.</p

Nuclear ISO6 and ISO12 FMRP are present in Cajal bodies.

<p>(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).</p

Effects of Leptomycin B on nuclear FMRP localization.

<p>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.</p

FMRP is present in the isolated nuclear fraction but not in nuclei.

<p>(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 <i>in situ</i>. 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.</p