FMRP shows a diverse subcellular distribution pattern in HeLa cells as revealed by subcellular fractionation analysis.

<p>(A) Experimental cell fractionation procedure employing several differential centrifugation steps. Cells were fractionated into six distinct fractions, including heavy membrane (plasma membrane and rough endoplasmic reticulum), light membrane (polysomes, golgi apparatus, smooth endoplasmic reticulum), cytoplasm (cytoplasm and lysosomes), enriched nuclear membrane (containing rough endoplasmic reticulum), nucleoplasm, and nucleoli. S, supernatant; P, pellet. (B) FMRP is largely absent in the cytoplasm and nucleoplasm and predominantly localizes to solid compartments. The protein concentrations were normalized in all fractions with exception of the nucleoplasm due its low protein content as compared to the other fractions. In each lane, 5 μg proteins were loaded except for the nucleoplasm, where one μg was used. In addition to FMRP and its binding partner CYFIP, the fractions were analyzed by using different subcellular marker, including Gα_q/11, Na⁺/K⁺-ATPase and Rac1 (plasma membrane), EEA2 (endosomes), GAPDH (cytoplasm), eIF5 and RPLP0 (ribosomes and rough ER). Nuclear markers included histone H3 and lamin B1. Nucleolin was used as nucleolar marker. (C) Detection of FMRP in mitochondria. The presence of FMRP in isolated mitochondrial fraction was analyzed by SDS-PAGE and immunoblotting, using antibodies against FMRP, two mitochondrial proteins MTCO2 and ACAT1, the cytosolic GAPDH as well as the nuclear proteins lamin B1, histone H3 and nucleolin. Equal protein amounts of the mitochondrial fraction and the total cell lysate were used.</p

FMRP and nucleolin interact in both cytosolic high molecular weight and nuclear low molecular weight complexes.

<p>(A, B) Native FMRP protein complexes were fractionated by loading the light membrane (A) and nucleolar (B) fractions on a superose 6 size exclusion chromatography column. The absorbance of the column eluent at 280 nm (A₂₈₀) was plotted against the elution volume (ml). Different FMRP binding partners and markers are shown, including histone H3 and GAPDH as negative controls in the endomembrane and nucleolar fractions, respectively. The elution positions of standard proteins employed include thyroglobuline (669 kDa), ferritin (440 kDa), aldolase (158 kDa), ovalbumin (75 kDa), carbonic anhydrase (29 kDa), ribonuclease (13.7 kDa), and aprotinin (6.5 kDa). The peak fractions, as indicated by a solid line, were subjected to SDS-PAGE and immunoblotting using antibodies against FMRP (71 kDa), nucleolin (76 kDa), CYFIP2 (146 kDa), RPLP0 (34 kDa) and eIF5 (58 kDa) LM, light membrane; Nu, nucleoli. The molecular mass of the peak fractions is indicated above the peaks. (C) Interaction of FMRP with CYFIP, nucleolin, eIF5, and RPLP0 as analyzed by co-immunoprecipitation. Endogenous FMRP was immunoprecipitated from HeLa cell lysates using an anti-FMRP antibody before and after RNase treatment. FMRP co-precipitated with nucleolin, RPLP0, eIF5, and CYFIP2. Interaction with the latter two proteins was sensitive to RNase treatment. Proteins were visualized by using antibodies against FMRP, nucleolin, eIF5, CYFIP2 and RPLP0. N-WASP and GAPDH were used as a negative IP controls. IP, immunoprecipitation; TCL, total cell lysate. (D) Direct interaction between FMRP and nucleolin. GST pull-down experiments were conducted by mixing bacterial lysate expressing His-tagged FMRP fl (upper panel) or FMRP Nterm (lower panel) with different GST-fused nucleolin proteins (RRM1&2, aa 284–466; RRM3&4, aa 467–644; RRM3&4-RGG, aa 499–710; RGG, aa 645–710) immobilized on GSH sepharose beads. Proteins retained on the beads were resolved by SDS-PAGE and processed for Western blot using a monoclonal antibody against FMRP. Mixed samples before performing pulldown (PD) analysis were used as input control. (E) Low-affinity interaction between the FMRP Nterm and the nucleolin RGG. Fluorescence polarization assay was used as a tool for monitoring the interaction of the FMRP Nterm (increasing concentrations as indicated) with the IAEDANS-labeled fluorescent RGG (0.5 μM) (open circles). As negative controls, FMRP Nterm was titrated into IAEDANS alone (0.5 μM) (closed circles). The inset depicts the displacement of FMRP Nterm from IAEDANS-labeled fluorescent RGG by increasing concentrations of unlabeled RGG and the synthetic peptide construct 5(KPR)TASP.</p

The C-terminal region of FMRP contains evolutionary conserved nucleolar localization signals.

<p>(A) Domain organization and motifs of FMRP. Schematic diagram of FMRP architectures highlights major domains and motifs. FCT, FMRP C-terminus; KH1 and KH2, tandem K (described first in the hnRNP K protein) homology domain; NES, nuclear export signal; NLS, nuclear localization signal; NoLS, nucleolar localization signal; PPID, protein-protein interaction domain; RGG, arginine-glycine-glycine region; P, phosphorylation sites; Tud1 and Tud2, tandem Tudor (also called Agenet) domains. The C-terminal region (Cterm; aa 444–632) of FMRP contains two NoLSs, identified in this study. Two further FRMP fragment used were Nterm (1–218) and a Central region (212–425). (B) Overexpression of the Cterm wild-type (wt) and its variants on HeLa cells. Cterm 1: QKKEK changed to EEEeE; Cterm 2: RRGDGRRR changed to EEgdgEEE; Cterm 3: RR changed to EE; Cterm 1+2: a combination of Cterm 1 and 2 mutations; Cterm 1+3: a combination of Cterm 1 and 3 mutations. Cterm 2 and Cterm 1+2 revealed a change in protein mobility (**) as compared to the wild-type and the other variants (*). (C) NoLS prediction of FMRP Cterm using the NoLS predictor program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091465#pone.0091465-Scott1" target="_blank">[58]</a>. Graph shows the probability of NoLS distribution (represented by score) plotted against the amino acid sequence of FMRP Cterm (444–632). Three motifs and critical positively charged residues are marked blue. (D) Multiple sequence alignment of the three predicted NoLS motifs 1, 2, and 3 of FMRP Cterm from different species (upper panel) as well as FMRP transcripts and homologous proteins (lower panel). Basic residues (blue), which are changed to glutamic acids (red) are highlighted. Upper panel: FMRP sequences from different species are human (accession number 544328), orangutan (197102198), rat (30794228), frog (53749722) and zebrafish (23308667). Lower panel: FMRP transcripts and homologous proteins are transcript 6 (297374777), 7 (297374779), 9 (297374791) and 12 (297374789) as well as FXR1P (61835148) and FXR2P (259013556). (E) Nucleolar localization of FMRP. cLSM images of HeLa cells transfected with FMRP fl, Nterm, Central, Cterm (wt) and Cterm variants (anti-flag; green channel) costained with endogenous nucleolin (anti-nucleolin; red channel) and DNA (DAPI; blue channel) revealed that Cterm (wt), (1), (2), (3) and (1+3) colocalize with nucleolin in the nucleolus. In contrast this colocalization was absent in the case of Cterm (1+2). Cytoplasmic distribution of FMRP Nterm and the subnuclear distribution of endogenous nucleolin are highlighted by arrows. Scale bar: 10 μm.</p

FMRP is localized at various intracellular sites in HeLa cells.

<p>Confocal laser scanning microscopy (cLSM) images of HeLa cells depicting endogenous FMRP (green channel) costained with various cytosolic (A) and nuclear (B) markers (red channel), including antibodies against CYFIP2, RPLP0 (ribosomal proteins), nucleolin (nucleolar marker), MTCO2 (mitochondrial protein), NUP62 (nucleoporins), lamin B1 (nuclear intermediate filament proteins), and calreticulin (endoplasmic reticulum marker). Detection of Na⁺/K⁺-ATPase and phalloidin staining were used to detect the cellular membrane and F-actin, respectively. DNA was stained by using DAPI (blue channel). Boxed areas in the merged panels depict enlarged areas of interest. Scale bar: 10 μm.</p

AUC-SV data for NPM1^FL, NPM1^OD, and US11^FL, respectively.

<p>MW, molecular weight; S20,w (S), sedimentation rate at 20°C; f/f0, frictional coefficient. In all three cases the values refer to a single, dominant species, which represented more than 90% of the sample.</p><p>AUC-SV data for NPM1^FL, NPM1^OD, and US11^FL, respectively.</p

Direct NPM1 interaction with HIV-1 Rev.

<p>(A) Qualitative interaction analysis by GST pull-down assay and subsequent CBB staining. NPM1 FL, OD and HRBD, but not RBD, displayed a selective interaction with HIV-1 Rev (upper panel), which was also observed after an RNase A treatment (lower panel). (B) Quantitative interaction analysis by ITC. The binding parameters for the interaction between NPM1^FL and Rev were obtained using ITC. Titration of NPM1^FL (750 μM) to Rev^FL (35 μM) showed an exothermic response (negative peaks) indicating that Rev selectively interacts with NPM1^FL. The upper graph shows calorimetric changes plotted versus the time and the lower graph represents the changes in temperature according to the molar ratio of the interacting proteins. (C) No interaction was observed in a control experiments by titrating NPM1^RBD (300 μM) to Rev^FL (30 μM).</p

NPM1 association with HSV-1 US11 in the cell.

<p>(A) Nucleolar colocalization of endogenous NPM1 with myc-US11. Confocal images of HeLa cells transfected with myc-US11 were obtained by staining endogenous NPM1 (Mouse anti-NPM1 (ab10530)), myc-US11 (anti-myc antibody), and filamentous actin (rhodamine-phalloidin). For clarity, a boxed area in the merged panel shows colocalization of NPM1 and US11 in the nucleolus as pointed by arrows. Scale bar: 20 μm. (B) Myc-US11 associates with endogenous NPM1 in COS-7 cells. NPM1 was co-immunoprecipitated with myc-US11 overexpressed in COS-7 cells using anti-myc antibody. A normal Rabbit IgG and sample without antibody were used as IP controls. Input, 5% of total cell lysate; IP, immunoprecipitation; IB, immunoblotting. (C) Myc-US11^FL displayed an interaction with NPM1^FL. Myc-US11^FL was pulled down with the GST-fusion NPM1^FL, but not with GST, which was used as a negative control. Samples prior pull-down (PD) analysis were used as input control.</p

The synthetic peptide CIGB-300 competes with Rev and US11 by binding NPM1^OD with high-affinity.

<p>(A) CIGB-300 consists of the cyclic P15 (blue) and the Tat (purple) peptides, and labeled with fluorescein (green; FITC). (B) Fluorescence polarization experiments conducted by titrating increasing amounts of NMP1 variants, Rev, US11, and GST to 0.1 μM FITC-labelled CIGB-300 (f CIGB-300). A high affinity interaction with the peptide was only observed for NPM1^FL and NPM1^OD, resulting from an increase of polarization, but not for Rev, US11, GST, and the other NPM1 variants. (C-D) Contrary to US11, Rev only displaced NPM1^OD from its fCIGB-300 complex. Displacement experiments were performed by adding increasing amounts of Rev or US11 to the NPM1^FL-fCIGB-300 complex (C) or to the NPM1^OD-fCIGB-300 complex (D). (E) A proposed NPM1^OD-CIGB-300 docking model of pentameric NPM1^OD structure in the complex with CIGB-300. Cyclic part (blue) and basic part (purple) of the peptide shown as sticks and ribbons wraps around several monomeric units of NPM1 represented by surfaces in different colors shown in top view (left), rotated orientation (middle), and the bottom view (right).</p

Physical interaction of HSV-1 US11 with NPM1.

<p>(A) C-terminal region of US11 largely contributes to NPM1 interaction. Pull-down experiments were conducted with purified proteins in the presence of RNase A by using GST-fused US11^FL, US11^Nterm, US11^Cterm, and GST as a negative control. For the detection of NPM1 variants two different antibodies were used, ab52644 recognized an N-terminal epitope containing in NPM1^FL and NPM1^OD, and ab10530 recognized a C-terminal epitope containing in NPM1^HRBD and NPM1^RBD. The same pattern of interaction was obtained for the N-terminal and the C-terminal parts of US11, although the interaction between NPM1^FL and NPM1^OD with US11^Nterm was much weaker than with US11^Cterm. The exposure time was 1 min for all the blots. (B-C) US11 binds NPM1 with a binding constant in the low micromolar range. To measure the binding parameter for the NPM1-US11 interaction, 1.2 mM NPM1^FL (B) and buffer (C) were titrated to 60 μM US11^FL. Both NPM1 and US11 were treated with RNase A. Conditions were the same as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0143634#pone.0143634.g002" target="_blank">Fig 2</a>. US11 binding to NPM1 is an endothermic reaction. (D) US11 binds to a pentameric NPM1. aSEC-MALS/RI analysis of NPM1^OD, US11^FL, and a mixture of both proteins revealed an oligomeric nature of NPM1^OD with a molecular weight (MW) of 66.1 kDa corresponding to the pentameric form. Obtained MW for US11 was 16.6 kDa, which matches the theoretical MW of 16.7 kDa for a monomeric US11 (upper panel). SDS-PAGE and CBB staining of the aSEC (Superdex 200, 10/300) elution fractions of NPM1^OD, US11^Fl, and a mixture of both clearly revealed a NPM1-US11 complex formation (lower panel). Both NPM1 and US11 were treated with RNase A. The MW of this complex corresponds to 76.6 kDa for a pentameric NPM1^OD, and a monomeric US11^FL. A MW of 21.8 kDa was measured that is estimated to an unbound US11^FL.</p

CIGB300 treatment interferes with HIV-1 production.

<p>CIGB-300 treated or untreated HOS.CD4.CXCR4 cells were infected with NL4.3 virus at an MOI of 1. Culture supernatant was collected 48 and 72 h post infection and virus titer was determined. The figure shows one representative experiment out of four, in which virus quantification was performed by TZM-bl cell titration. Values are the means ± S.D. of three measurements. Statistical significance (P) was calculated by the Student`s t-test: ***P<0.002; **P<0.02.</p