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

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 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

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