PubMed Health⌕ Search

Biomedical subjects

F Amalric

Publications and source records attributed to F Amalric.

At least 37 records · Page 2Linked to original sources
PubMed1997

Two RNA-binding domains determine the RNA-binding specificity of nucleolin.

Nucleolin is an abundant nucleolar RNA-binding protein that seems to be involved in many aspects of ribosome biogenesis. Nucleolin contains four copies of a consensus RNA-binding domain (CS-RBD) found in several other proteins. In vitro RNA-binding studies previously determined that nucleolin interacts specifically with a short RNA stem-loop structure. Taken individually, none of the four CS-RBDs interacts significantly with the RNA target, but a peptide that contains the first two adjacent CS-RBDs (R12) is sufficient to account for nucleolin RNA-binding specificity and affinity. The full integrity of these two domains is required, since N- or C-terminal deletion abolishes the specific interaction with the RNA. Mutation of conserved amino acids within the RNP-1 sequence of CS-RBD 1 or 2 drastically reduces the interaction with the RNA, whereas mutation of the analogous residues in CS-RBDs 3 and 4 has no effect in the context of the R1234G protein (which corresponds to the C-terminal end of nucleolin). Our results demonstrate that nucleolin RNA-binding specificity is the result of a cooperation between two CS-RBDs (RBDs 1 and 2) and also suggests a direct or indirect involvement of the RNP-1 consensus sequence of both CS-RBDs in the recognition of the RNA target.

Animals↗
PubMed1996

Fibroblast growth factor-2 binds to the regulatory beta subunit of CK2 and directly stimulates CK2 activity toward nucleolin.

The presence of fibroblast growth factor-2 (FGF-2) in the nucleus has now been reported both in vitro and in vivo, but its nuclear functions are unknown. Here, we show that FGF-2 added to nuclear extract binds to protein kinase CK2 and nucleolin, a CK2 natural substrate. Added to baculovirus-infected cell extracts overexpressing CK2 or its isolated subunits, FGF-2 binds to the enzyme through its regulatory beta subunit. Using purified proteins, FGF-2 is shown to directly interact with CK2 and to stimulate CK2 activity toward nucleolin. Furthermore, a mitogenic-deficient FGF-2 mutant protein has an impaired ability to interact with CK2 and to stimulate CK2 activity using nucleolin as substrate. We propose that in growing cells, one function of nuclear FGF-2 is to modulate CK2 activity through binding to its regulatory beta subunit.

Animals↗
PubMed1996

Nucleolin is a sequence-specific RNA-binding protein: characterization of targets on pre-ribosomal RNA.

Nucleolin is an abundant nucleolar protein, which plays an essential, but largely unknown role in ribosome biogenesis. Nucleolin contains four consensus RNA-binding domains (CS-RBD), the presence of which suggests that the molecular function of this protein is likely reflected by its RNA-binding properties. Indeed, by immunocytological analysis performed on ribosomal transcription units, we have found several nucleolin molecules associated with nascent pre-rRNA. In mouse, two high-affinity binding sites with an apparent dissociation constant (Kd) of 50 to 100 nM have been mapped in the 5' ETS upstream from the early pre-rRNA processing site. Interestingly, nucleolin of mouse origin has recognized analogous sequences in the 5' ETS of human pre-rRNA. In parallel, selection-amplification (SELEX) experiments have identified an 18-nucleotide long RNA sequence that binds nucleolin with high affinity (Kd 5 to 20 nM) and shares a common UCCCGA motif with the characterized pre-rRNA binding sites. By mutagenesis and a structural analysis, we have characterized the nucleolin RNA binding site and found that it is constituted by a minimal 18-nucleotide long stem-loop structure. The sequence UCCCGA that is found within the hairpin loop is necessary for the specific interaction. Mutation of any of the C or G residues within this motif abolishes nucleolin interaction. Furthermore, point mutation in the stem that completely disrupt the hairpin structure also prevents nucleolin binding. By determining the minimal 5' and 3' ends of the RNA that is bound to the protein we concluded that nucleolin binding site is constituted by a short four to five-base-pair stem and an eight-nucleotide loop. This structural motif is very similar to hairpins recognized by two other CS-RBD-containing proteins (U1 snRNP A and U2 snRNP B"). Possible functional implications of our findings are discussed.

Animals↗
PubMed1996

Localization of nucleolin binding sites on human and mouse pre-ribosomal RNA.

Nucleolin, a major RNA binding protein of the nucleolus is found associated mainly to the pre-ribosomal particles and is absent from the cytoplasmic mature ribosomes. The role of this protein in ribosome biogenesis remains largely unknown, and is likely to be reflected by its RNA binding properties. Nucleolin contains in its central domain four RNA recognition motifs (RRM, also called RBD for RNA binding domain) which are conserved among different species. RNA binding studies have revealed that nucleolin interacts specifically with a short stem loop structure called NRE (nucleolin recognition element). We show that nucleolin extracted from human, hamster and mouse cells interacts with the same specificity and affinity to a mouse 5'ETS (external transcribed spacer) RNA fragment which contains a NRE motif. A similar structure within the human 5'ETS is also efficiently recognized by mouse nucleolin. We identified putative NRE not only in the 5'ETS but also in the 3'ETS, ITS (internal transcribed spacer) and in the 18S and 28S RNA sequences. This is in agreement with in vivo cross-linking data and a previous immunocytological analysis of ribosomal transcription units. Interestingly, we found that all the NRE localized in the 28S region are within the variable domains. Despite considerable sequence divergence of these domains, several of the NRE have sequences perfectly conserved between these two species. This suggests that these nucleolin binding sites might be functionally important, in particular for ribosome biogenesis.

Animals↗
PubMed1996

Translation of CUG- but not AUG-initiated forms of human fibroblast growth factor 2 is activated in transformed and stressed cells.

Four isoforms of the human fibroblast growth factor 2 (FGF-2), with different intracellular localizations and distinct effects on cell phenotype, result from alternative initiations of translation at three CUG and one AUG start codons. We showed here by Western immunoblotting and immunoprecipitation that the CUG-initiated forms of FGF-2 were synthesized in transformed cells, whereas "normal" cells almost exclusively produced the AUG-initiated form. CUG-initiated FGF-2 was induced in primary skin fibroblasts in response to heat shock and oxidative stress. In transformed cells and in stressed fibroblasts, CUG expression was dependent on cis-elements within the 5' region of FGF-2 mRNA and was not correlated to mRNA level, indicating a translational regulation. UV cross-linking experiments revealed that CUG expression was linked to the binding of several cellular proteins to FGF-2 mRNA 5' region. Since translation of FGF-2 mRNA was previously shown to occur by internal ribosome entry, a nonclassical mechanism already described for picornaviruses, the cross-linking patterns of FGF-2 and picornavirus mRNAs were compared. Comigration of several proteins, including a p60, was observed. However, this p60 was shown to be different from the p57/PTB internal entry factor, suggesting a specificity towards FGF-2 mRNA. We report here a process of translational activation of the FGF-2 CUG-initiated forms in direct relation with trans-acting factors specific to transformed and stressed cells. These data favor a critical role of CUG-initiated FGF-2 in cell transformation and in the stress response.

Animals↗
PubMed1995

Alternative translation initiation of the Moloney murine leukemia virus mRNA controlled by internal ribosome entry involving the p57/PTB splicing factor.

Moloney murine leukemia virus (Mo-MuLV) genomic mRNA codes for two gag precursors by alternative initiations of translation. An AUG codon governs the synthesis of the retroviral capsid proteins precursor, whereas a CUG codon directs the synthesis of a glycosylated cell surface antigen, the gross cell surface antigen. Control of the relative synthesis of the two precursors is crucial for MuLV infectivity and pathology. Furthermore, the MuLV mRNA leader sequence is very long and should inhibit translation according to the classical scanning model. This suggests a different translation initiation mechanism allowing gag efficient expression. We demonstrate, by using bicistronic vectors expressed in COS-7 cells, that the Mo-MuLV mRNA leader drives translation initiation by internal ribosome entry. We have localized the internal ribosome entry site (IRES) between the two initiation codons. This 126 nucleotide long IRES implies an oligopyrimidine tract located 45 nucleotides upstream of AUG codon. UV cross-linking and affinity chromatography experiments show that the PTB/p57 splicing factor specifically interacts with this oligopyrimidine tract. The MuLV IRES controls alternative translation initiation by activating the capsid protein precursor expression. This gag translational enhancer could exist in other retroviruses.

Animals↗
PubMed1995

Human DNA helicase IV is nucleolin, an RNA helicase modulated by phosphorylation.

The cDNA encoding human DNA helicase IV (HDH IV), a 100-kDa protein which unwinds DNA in the 5' to 3' direction with respect to the bound strand, was cloned and sequenced. It was found to be identical to the human cDNA encoding nucleolin, a ubiquitous eukaryotic protein essential for pre-ribosome assembly. HDH IV/nucleolin can unwind RNA-RNA duplexes, as well as DNA-DNA and DNA-RNA duplexes. Phosphorylation of HDH IV/nucleolin by cdc2 kinase and casein kinase II enhanced its unwinding activity in an additive way. The Gly-rich C-terminal domain possesses a limited ATP-dependent duplex-unwinding activity which contributes to the helicase activity of HDH IV/nucleolin.

Adenosine Triphosphatases↗
PubMed1995

Alternative translation of human fibroblast growth factor 2 mRNA occurs by internal entry of ribosomes.

Alternative initiations of translation of the human fibroblast growth factor 2 (FGF-2) mRNA, at three CUG start codons and one AUG start codon, result in the synthesis of four isoforms of FGF-2. This process has important consequences on the fate of FGF-2: the CUG-initiated products are nuclear and their constitutive expression is able to induce cell immortalization, whereas the AUG-initiated product, mostly cytoplasmic, can generate cell transformation. Thus, the different isoforms probably have distinct targets in the cell. We show here that translation initiation of the FGF-2 mRNA breaks the rule of the cap-dependent ribosome scanning mechanism. First, translation of the FGF-2 mRNA was shown to be cap independent in vitro. This cap-independent translation required a sequence located between nucleotides (nt) 192 and 256 from the 5' end of the 318-nt-long 5' untranslated region. Second, expression of bicistronic vectors in COS-7 cells indicated that the FGF-2 mRNA is translated through a process of internal ribosome entry mediated by the mRNA leader sequence. By introducing additional AUG codons into the RNA leader sequence, we localized an internal ribosome entry site to between nt 154 and 318 of the 5' untranslated region, just upstream of the first CUG. The presence of an internal ribosome entry site in the FGF-2 mRNA suggests that the process of internal translation initiation, by controlling the expression of a growth factor, could have a crucial role in the control of cell proliferation and differentiation.

Animals↗
PubMed1995

[Conservative treatment of sub-clinical noninfiltrating cancers of the breast (102 EOA in situ, TONO)].

102 patients with sub-clinical intra ductal non invasive breast cancer (T0N0) treated by limited surgery and curative radiation therapy. Follow-up ranged from 2 to 10 years with a median follow-up of 59 months. The long term survival rate of this therapeutic approach, consisting of simple excision without any adjuvant treatment and radical mastectomy was close to 100%. The actuarial local recurrence rate was only 8.6% at 10 years (confidence interval: 4.6 to 12.6%), which strongly suggests that radiation therapy is active on multicentric foci. Salvage surgery could be performed in every case of local recurrence. The survival rate of in situ breast cancer (T0N0) treated by conservative radio-surgery is comparable to that of radical mastectomy, while cosmetic results and psychological impact appear to be better for the conservative technique (breast preservation rate = 91 to 96%).

Breast Neoplasms↗
PubMed1995

Basic fibroblast growth factor (FGF-2) internalization through the heparan sulfate proteoglycans-mediated pathway: an ultrastructural approach.

Biochemical studies have shown that basic fibroblast growth factor (bFGF or FGF-2) is internalized by two pathways, after binding to either FGF tyrosine kinase receptors or to heparan sulfate proteoglycans (HSPG). To get insights on the HSPG-mediated pathway, we have examined by electron microscopy the intracellular route of bFGF-HRP, a monovalent conjugate of bFGF and horseradish peroxidase which was found to bind to HSPG only and was detectable by electron microscopy. bFGF-HRP association to adult bovine aortic endothelial (ABAE) cells or baby hamster kidney (BHK) cells was inhibited by a high molar excess of native bFGF, a 2 M NaCl wash at neutral pH, heparin and heparan sulfate, but not by chondroitin 4-sulfate or chondroitin 6-sulfate. bFGF-HRP was not able to displace [125I]bFGF from its high-affinity binding sites, and the dissociation constant of its binding to ABAE cells was estimated at 3 nM. Time-course experiments were performed to follow bFGF-HRP endocytosis in ABAE cells. bFGF-HRP was found to enter the cell after binding to the plasma membrane or extracellular matrix. On the cell surface, the probe accumulated in noncoated flask-shaped invaginations and in caveolae rather than in clathrin-coated pits. Immediately after endocytosis, bFGF-HRP was detected in pleiomorphic tubulovesicular and tubulocisternal early endosomes. Multivesicular bodies contained diaminobenzidine (DAB) precipitate after 5 to 15 min, but lysosomes were not labeled before 1 h, indicating a delayed transfer from late endosomes to lysosomes. Labeling was never detected in the nucleus, even after intensification of the DAB reaction product by silver-gold enhancement. Similar endocytic pathways and intracellular locations were observed in other endothelial and non-endothelial cell types. These results suggest that bFGF associated to HSPG can enter the cell via several pathways and follows mainly a degradative route.

Animals↗
PubMed1995

Expression of basic fibroblast growth factor (bFGF) and FGF-receptors in human leukemic cells.

Recent reports have suggested that basic fibroblast growth factor (bFGF) could play a permissive role in hematopoiesis, in combination with specific colony-stimulating factors. We investigated the expression of bFGF and FGF-receptors (FGF-Rs) in leukemic cell lines of various hematopoietic lineages. Three protein isoforms of bFGF of approximately 18, 22 and 24 kDa were detected in the myeloid cell line K562, but not in myelomonocytic or lymphoid (T or B) cell lines. In vitro-induced differentiation of K562 cells did not change the pattern of expression of the different bFGF isoforms. Accordingly, the mRNA of bFGF was found expressed in K562, but not in other leukemic lines tested, as assayed by reverse transcript amplification (RT-PCR). Using the same technique, we searched for the presence of high affinity FGF-Rs on these cells: in eight out of ten cell lines tested, mRNA for at least one FGF-R among FGF-R1, FGF-R3 or FGF-R4 was expressed, whereas FGF-R2 was never detected. We found that two cell lines were responsive to bFGF in different biological assays: (i) in K562 myeloid cells induced to differentiate by hemin, preincubation with bFGF and heparin increased cell viability and decreased hemin-induced DNA fragmentation, without affecting erythroid differentiation; and (ii) in U937 monocytic cells, the production of plasminogen activator was increased by bFGF or aFGF in combination with heparin. Binding experiments with 125I-bFGF (up to 200 pM) in the presence of heparin revealed high affinity receptors on the K562 and U937 cell lines (1177 +/- 440 and 392 +/- 184 sites/cell, Kd = 61.7 +/- 8.6 and 43.1 +/- 13.5 pM, respectively). Thus our results strongly suggest that cells of hematopoietic origin could express functional FGF-receptors.

Base Sequence↗
PubMed1994

Purification and characterization of the 210-amino acid recombinant basic fibroblast growth factor form (FGF-2).

Four forms of basic fibroblast-growth factor (bFGF or FGF-2) using one AUG (155 amino acids) and three upstream CUG (210, 201 and 196 amino acids) start codons, were synthesized through an alternative use of initiation codons. The 210-amino acid form of FGF-2 (210FGF-2) was expressed in a plasmid vector under the control of a bacteriophage T7 RNA polymerase promoter system in Escherichia coli. Characterization of the purified protein was performed by electrospray mass spectrometry and Edman degradation. The recombinant 210FGF-2 produced in E. coli had a mitogenic activity similar to the 146-amino acid form extracted from tissues.

Amino Acid Sequence↗
PubMed1994

Identification of a segment of the small nucleolar ribonucleoprotein-associated protein GAR1 that is sufficient for nucleolar accumulation.

GAR1 is a 25-kDa nucleolar protein that is essential for yeast cell growth. The protein is associated with a subset of small nucleolar RNAs and is required for pre-rRNA processing. By expressing in yeast various deletions of GAR1 fused to a reporter protein, we have searched for which particular domain of GAR1 can account for its nucleolar localization. We report here that the glycine/arginine-rich domains of GAR1, which are shared by several other nucleolar proteins, are neither sufficient nor required for the steady-state accumulation of the fusion protein in the nucleolus. We further demonstrate that the central domain of GAR1 is both sufficient to target the beta-galactosidase to the yeast nucleolus and to restore the growth of a strain deficient in GAR1. As opposed to the other characterized nucleolar proteins, the nucleolar targeting domain of GAR1 does not exhibit any homology with the SV40 T-antigen-type nuclear localization sequence. Moreover, none of the modified GAR1 proteins that we examined has allowed us to distinguish the nuclear and nucleolar targeting domains. The presence in GAR1 of a single domain that is responsible for both nuclear entry and nucleolar accumulation suggests that GAR1 either could be carried piggyback by another nucleolar component, possibly as part of a small nucleolar ribonucleoprotein particle, or could be transported to the nucleolus by using a pathway different from the other nucleolar proteins.

Amino Acid Sequence↗
PubMed1994

Fibroblast growth factor-2 (FGF-2) in the nucleus: translocation process and targets.

FGF-2 (basic fibroblast growth factor) was recently detected in the nucleus of a variety of cell types. The large isoforms contain a functional nuclear localization signal that allows their nuclear accumulation in producing cells, while a small amount of FGF-2 added exogenously to target cells is translocated to the nucleus in phase G1 of the cell cycle according to an unknown process. We report here using Chinese hamster ovary cell mutants bearing deficiency in heparan sulfate proteoglycans (HSPGs) synthesis that HSPGs are required for transport of exogenous FGF-2 to the nucleus. Furthermore a co-transport was suggested since an active complex containing FGF-2 and HSPGs was isolated from nuclei of treated cells. Several FGF-2 nuclear targets were described. In vivo as in vitro, it activates rDNA transcription and it binds to a specific DNA sequence that is present in the non-transcribed spacer of ribosomal genes. In vitro, FGF-2 has a strong affinity for histone H1 and it activates the protein kinase CKII. In the nucleus FGF-2 could regulate gene expression through modulation of chromatin structure.

Animals↗
PubMed1994

Is the coiled body involved in nucleolar functions?

Coiled bodies (CBs) are structural constituents observed in nuclei of most eukaryotic cells. They usually occur in the nucleoplasm as well as in contact with the nucleolar surface. In this work we studied the hepatocyte nuclei of hibernating dormice in order to investigate possible modifications of CBs along the seasonal cycle. CBs were abundant during hibernation and rapidly disappeared upon arousal from hibernation. Moreover, CBs were frequently found to be integrated into the nucleolar body. Immunocytochemical analyses showed that CBs contain nucleoplasmic as well as nucleolar RNA-processing factors, suggesting an "ambiguous" role for this organelle in the nuclear functions.

Animals↗
PubMed1994

Physiologically inactive nucleoli contain nucleoplasmic ribonucleoproteins: immunoelectron microscopy of mouse spermatids and early embryos.

Physiologically inactivating or reactivating nucleoli represent a good model to investigate modifications of the nucleolar activity, which influence to a great extent the morphology of this organelle. We have studied the nucleoli of mouse spermatids during gradual inactivation, as well as those of early mouse preimplantation embryos, which undergo reactivation. In the 2-cell systems, inactive nucleoli are represented by homogeneously, finely fibrillar spherical bodies. With the aim of clarifying the composition of these nucleoli, we have analyzed them by means of immunoelectron microscopy using specific antibodies directed against nucleoplasmic snRNPs, hnRNPs, ribosomal proteins, or fibrillarin as well as by cytochemical methods for visualizing DNA and RNA. Our results indicate that RNA is present in zygote and 2-cell nucleolus precursor bodies (NPBs) as well as in 4- to 8-cell or more advanced embryo nucleoli, but not in inactive spermatid nucleoli. DNA is absent from inactive spermatid nucleoli and NPBs but is present within the nucleolus-associated chromatin and the nucleolonema of active nucleoli. The dense masses constituting the NPBs of the zygote and 2-cell embryos contain ribosomal proteins and fibrillarin but also hnRNPs and nucleoplasmic snRNPs. Of these, only fibrillarin is present in the spermatid residual nucleoli.

Animals↗
PubMed1994

Heparan sulfate proteoglycans as transducers of FGF-2 signalling.

The fibroblast growth factor-2 (FGF-2) low-affinity binding sites, heparan sulfate proteoglycans (HSPGs), function as modulators of FGF-2 activity. It is noteworthy that HSPG binding protects FGF-2 from denaturation and proteolytic degradation, provides a matrix-bound or cell-surface reservoir of this factor for the cells and is required for the activation of FGF high-affinity receptors. In our study we investigated the biological meaning of FGF-2 internalization mediated through its low-affinity binding sites, HSPGs. Using as model system L6 myoblasts lacking endogenous FGF receptors (FGFRs), we demonstrated that these cells internalize FGF-2 efficiently through an HSPG-mediated pathway. FGF-2 internalization occurring through HSPGs was paralleled by an increase in the activity of urokinase plasminogen activator (u-PA). The u-PA-inducing activity of FGF-2 was strictly correlated to its internalization, as chlorate treatment, which causes a strong inhibition of FGF-2 internalization, abrogated the u-PA-inducing activity of FGF-2. In addition, expression of functional FGF high-affinity receptors (FGFR-1) did not enhance u-PA in L6 myoblasts upon FGF-2 stimulation. According to our results we propose that FGF-2 internalization mediated through HSPGs may transduce FGF-2 signalling such as u-PA-activity stimulation. Thus, HSPGs may act as direct transducers of FGF signalling and indeed, different FGF-signalling pathways must exist.

3T3 Cells↗