François Amalric

Molecular Characterization of NF-HEV, a Nuclear Factor Preferentially Expressed in Human High Endothelial Venules

Lymphocyte homing to secondary lymphoid tissue and lesions of chronic inflammation is directed by multi-step interactions between the circulating cells and the specialized endothelium of high endothelial venules (HEVs). In this study, we used the PCR-based method of suppression subtractive hybridization (SSH) to identify novel HEV genes by comparing freshly purified HEV endothelial cells (HEVECs) with nasal polyp-derived microvascular endothelial cells (PMECs). By this approach, we cloned the first nuclear factor preferentially expressed in HEVECs, designated nuclear factor from HEVs (NF-HEV). Virtual Northern and Western blot analyses showed strong expression of NF-HEV in HEVECs, compared to human umbilical vein endothelial cells (HUVECs) and PMECs. In situ hybridization and immunohistochemistry revealed that NF-HEV mRNA and protein are expressed at high levels and rather selectively by HEVECs in human tonsils, Peyerss patches, and lymph nodes. The NF-HEV protein was found to contain a bipartite nuclear localization signal, and was targeted to the nucleus when ectopically expressed in HUVECs and HeLa cells. Furthermore, endogenous NF-HEV was found in situ to be confined to the nucleus of tonsillar HEVECs. Finally, threading and molecular modeling studies suggested that the amino-terminal part of NF-HEV (aa 1-60) corresponds to a novel homeodomain-like Helix-Turn-Helix (HTH) DNA-binding domain. Similarly to the atypical homeodomain transcription factor Prox-1, which plays a critical role in the induction of the lymphatic endothelium phenotype, NF-HEV may be one of the key nuclear factors that controls the specialized HEV phenotype.

Alternative initiation of translation determines cytoplasmic or nuclear localization of basic fibroblast growth factor.

Three forms of basic fibroblast growth factor (bFGF), initiated at an AUG (18 kDa) and two CUG (21 and 22.5 kDa) start codons, were produced following transfection of COS cells with human hepatoma bFGF cDNA. The subcellular localization of the different forms was investigated directly or by using chimeric genes constructed by fusion of the bFGF and chloramphenicol acetyltransferase open reading frames. The AUG-initiated proteins were cytoplasmic, while the CUG-initiated forms were nuclear. The signal sequence responsible for the nuclear localization of bFGF is contained within 37 amino acid residues between the second CUG and the AUG start codons. Alternative initiation of translation regulates the subcellular localization of bFGF and thus could modulate its role in cell growth and differentiation control.

Translocation of bFGF to the nucleus is G1 phase cell cycle specific in bovine aortic endothelial cells.

Primary cultures of adult bovine aortic endothelial (ABAE) cells require bFGF to grow. G1‐arrested cells, obtained after 48 h without serum and bFGF, were found to enter S phase and grow synchronously for at least two generations on addition of bFGF. In growing cells bFGF was detected both in the cytoplasm (90%) and in the nucleus (10%) where it accumulates in the nucleolus. It was not detected in the nucleus of confluent cells. bFGF uptake was continuous in the cytoplasm throughout the cell cycle with a maximum in G2, while nuclear uptake occurred only in late G1. Cytoplasmic bFGF (18.4 kd) is cleaved into a 16.5 kd peptide in G1 (t1/2 = 30 min). In the nucleus the 18.4 kd form was the only one detected 2 h following bFGF addition and was then cleaved into the 16.5 kd in early S phase. These results are consistent with the possibility that in addition to the classical pathway of signal transduction, bFGF is directly translocated to the nucleus in late G1, and could play a role in replication and/or in transcription of rDNA.

Nucleolin functions in the first step of ribosomal RNA processing

The first processing step of precursor ribosomal RNA (pre‐rRNA) involves a cleavage within the 5′ external transcribed spacer. This processing requires sequences downstream of the cleavage site which are perfectly conserved among human, mouse and Xenopus and also several small nucleolar RNAs (snoRNAs): U3, U14, U17 and E3. In this study, we show that nucleolin, one of the major RNA‐binding proteins of the nucleolus, is involved in the early cleavage of pre‐rRNA. Nucleolin interacts with the pre‐rRNA substrate, and we demonstrate that this interaction is required for the processing reaction in vitro. Furthermore, we show that nucleolin interacts with the U3 snoRNP. Increased levels of nucleolin, in the presence of the U3 snoRNA, activate the processing activity of a S100 cell extract. Our results suggest that the interaction of nucleolin with the pre‐rRNA substrate might be a limiting step in the primary processing reaction. Nucleolin is the first identified metazoan proteinaceous factor that interacts directly with the rRNA substrate and that is required for the processing reaction. Potential roles for nucleolin in the primary processing reaction and in ribosome biogenesis are discussed.

GAR1 is an essential small nucleolar RNP protein required for pre-rRNA processing in yeast.

Among the few proteins of the eukaryotic nucleolus that have been characterized, four proteins, nucleolin, fibrillarin, SSB1 and NSR1, possess a common structural motif, the GAR domain, which is rich in glycine and arginine residues. In order to examine whether the presence of this domain is characteristic of a family of nucleolar proteins, we investigated whether other yeast genes encode proteins containing GAR domains. We report here the sequence and the characterization of a new yeast gene, GAR1, which encodes a protein of 205 residues containing two GAR domains. GAR1 is a non‐ribosomal protein, localized in the yeast nucleolus, which is essential for cell growth. Immunoprecipitation with anti‐GAR1 antibodies shows that GAR1 is associated with a subset of snoRNAs, including snR10 and snR30. Depletion of GAR1 by expression under the control of a regulated GAL promoter, impairs processing of the 35S primary transcript of pre‐rRNA and prevents synthesis of 18S rRNA. GAR1 is thus the fifth member of a family of nucleolar proteins containing GAR domains, and is involved in rRNA metabolism.

Nucleolin Interacts with Several Ribosomal Proteins through Its RGG Domain

Nucleolin is one of the major nonribosomal proteins of the nucleolus. Through its four RNA-binding domains, nucleolin interacts specifically with pre-rRNA as soon as synthesis begins, but it is not found in mature cytoplasmic ribosomes. Nucleolin is able to shuttle between the cytoplasm and the nucleus. These data suggest that nucleolin might be involved in the nucleolar import of cytoplasmic components and in the assembly of pre-ribosomal particles. Here we show, using two-dimensional blots in a ligand blotting assay, that nucleolin interacts with 18 ribosomal proteins from rat (14 and 4 from the large and small subunit, respectively). The C-terminal domain of nucleolin (p50) interacts with 10 of these identified ribosomal proteins. In vitro binding assays show that the glycine-arginine rich domain of nucleolin (RGG domain) is sufficient for the interaction with one of these proteins. Interestingly, most of the proteins that interact with p50 belong to the core ribosomal proteins, which are resistant to extraction with high salt concentration. These findings suggest that nucleolin might be involved in the nucleolar targeting of some ribosomal proteins and in their assembly within pre-ribosomal particles.

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 β 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 β subunit.

THAP1 is a nuclear proapoptotic factor that links prostate-apoptosis-response-4 (Par-4) to PML nuclear bodies

Promyelocytic leukemia (PML) nuclear bodies (PML NBs) are discrete subnuclear domains organized by the promyelocytic leukemia protein PML, a tumor suppressor essential for multiple apoptotic pathways. We have recently described a novel family of cellular factors, the THAP proteins, characterized by the presence at their amino-terminus of an evolutionary conserved putative DNA-binding motif, designated THAP domain. Here, we report that THAP1 is a novel nuclear proapoptotic factor associated with PML NBs, which potentiates both serum withdrawal- and TNFα-induced apoptosis, and interacts with prostate-apoptosis-response-4 (Par-4), a well characterized proapoptotic factor, previously linked to prostate cancer and neurodegenerative diseases. We show that endogenous Par-4 colocalizes with ectopic THAP1 within PML NBs in primary endothelial cells and fibroblasts. In addition, we found that Par-4 is a component of PML NBs in blood vessels, a major site of PML expression in vivo. Finally, we investigated the role of the THAP domain in THAP1 activities and found that this putative DNA-binding domain is not required for Par-4 binding and localization within PML NBs, but is essential for THAP1 proapoptotic activity. Together, our results provide an unexpected link between a nuclear factor of the THAP family, the proapoptotic protein Par-4 and PML nuclear bodies.

Mitosis-specific phosphorylation of nucleolin by p34cdc2 protein kinase.

Nucleolin is a ubiquitous multifunctional protein involved in preribosome assembly and associated with both nucleolar chromatin in interphase and nucleolar organizer regions on metaphasic chromosomes in mitosis. Extensive nucleolin phosphorylation by a casein kinase (CKII) occurs on serine in growing cells. Here we report that while CKII phosphorylation is achieved in interphase, threonine phosphorylation occurs during mitosis. We provide evidence that this type of in vivo phosphorylation involves a mammalian homolog of the cell cycle control Cdc2 kinase. In vitro M-phase H1 kinase from starfish oocytes phosphorylated threonines in a TPXK motif present nine times in the amino-terminal part of the protein. The same sites which matched the p34cdc2 consensus phosphorylation sequence were used in vivo during mitosis. We propose that successive Cdc2 and CKII phosphorylation could modulate nucleolin function in controlling cell cycle-dependent nucleolar function and organization. Our results, along with previous studies, suggest that while serine phosphorylation is related to nucleolin function in the control of rDNA transcription, threonine phosphorylation is linked to mitotic reorganization of nucleolar chromatin.

Molecular cloning of Xenopus fibrillarin, a conserved U3 small nuclear ribonucleoprotein recognized by antisera from humans with autoimmune disease

Autoantibodies against U3 small nuclear ribonucleoprotein are associated with scleroderma autoimmune disease. They were shown to react with fibrillarin, a 34- to 36-kilodalton protein that has been detected in all eukaryotes tested from humans to yeasts. We isolated a 1.6-kilobase cDNA encoding fibrillarin from a Xenopus laevis cDNA library. The protein contains a 79-residue-long Gly-Arg-rich domain in its N-terminal region and a putative RNA-binding domain with ribonucleoprotein consensus sequence in its central portion. This is the first report of cloning of fibrillarin, and the deduced protein sequence is in agreement with the involvement of the protein in a ribonucleoprotein particle.