PubMed Health⌕ Search

Biomedical subjects

Danièle Hernandez-Verdun

Publications and source records attributed to Danièle Hernandez-Verdun.

14 recordsLinked to original sources
PubMed2006

A B23-interacting sequence as a tool to visualize protein interactions in a cellular context.

We report the characterization of a nucleolar localization sequence (NoLS) that targets the green fluorescent protein (GFP) into the granular component (GC) of nucleoli. This NoLS interacts in vitro specifically and directly with the major nucleolar protein B23 and more precisely with the region of B23 including the two acidic stretches. The affinity of NoLS for B23 is stronger than that of the HIV-1 Rev protein in vitro. Moreover, B23-NoLS interaction also occurs in vivo. Indeed, (1) NoLS confers on the GFP the behavior of B23 throughout the cell cycle, (2) the GFP-NoLS fusion and B23 remain colocalized after drug treatments, (3) a selective delocalization of B23 from nucleoli to nucleoplasm induces a concomitent delocalization of the GFP-NoLS fusion, and (4) the fusion of NoLS to fibrillarin makes it possible to colocalize fibrillarin and B23. Interestingly, by fusing NoLS to fibrillarin, both fibrillarin and the fibrillarin partner Nop56 are mislocalized in the GC of nucleoli. Similarly, by fusing the NoLS to MafG, part of the nuclear transcription factor NF-E2 composed of both MafG and p45 NF-E2, NF-E2 is redirected from the nucleoplasm to the nucleoli. Thus, we propose that the NoLS may be used as a tool to visualize and prove protein interactions in a cellular context.

Animals↗
PubMed2006

Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function.

BACKGROUND: The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv) has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. RESULTS: Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. CONCLUSION: The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv.

Active Transport, Cell Nucleus↗
PubMed2006

The nucleolus: a model for the organization of nuclear functions.

Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins. The size and organization of the nucleolus are directly related to ribosome production. The organization of the nucleolus reveals the functional compartmentation of the nucleolar machineries that depends on nucleolar activity. When this activity is blocked, disrupted or impossible, the nucleolar proteins have the capacity to interact independently of the processing activity. In addition, nucleoli are dynamic structures in which nucleolar proteins rapidly associate and dissociate with nucleolar components in continuous exchanges with the nucleoplasm. At the time of nucleolar assembly, the processing machineries are recruited in a regulated manner in time and space, controlled by different kinases and form intermediate structures, the prenucleolar bodies. The participation of stable pre-rRNAs in nucleolar assembly was demonstrated after mitosis and during development but this is an intriguing observation since the role of these pre-rRNAs is presently unknown. A brief report on the nucleolus and diseases is proposed as well as of nucleolar functions different from ribosome biogenesis.

Cell Cycle↗
PubMed2006

Compartmentation of the nucleolar processing proteins in the granular component is a CK2-driven process.

To analyze the compartmentation of nucleolar protein complexes, the mechanisms controlling targeting of nucleolar processing proteins onto rRNA transcription sites has been investigated. We studied the reversible disconnection of transcripts and processing proteins using digitonin-permeabilized cells in assays capable of promoting nucleolar reorganization. The assays show that the dynamics of nucleolar reformation is ATP/GTP-dependent, sensitive to temperature, and CK2-driven. We further demonstrate the role of CK2 on the rRNA-processing protein B23. Mutation of the major CK2 site on B23 induces reorganization of nucleolar components that separate from each other. This was confirmed in assays using extracts containing B23 mutated in the CK2-binding sites. We propose that phosphorylation controls the compartmentation of the rRNA-processing proteins and that CK2 is involved in this process.

Adenosine Triphosphate↗
PubMed2005

Nucleolus: from structure to dynamics.

The nucleolus, a large nuclear domain, is the ribosome factory of the cells. Ribosomal RNAs are synthesized, processed and assembled with ribosomal proteins in the nucleolus, and the ribosome subunits are then transported to the cytoplasm. In this review, the structural organization of the nucleolus and the dynamics of the nucleolar proteins are discussed in an attempt to link both information. By electron microscopy, three main nucleolar components corresponding to different steps of ribosome biogenesis are identified and the nucleolar organization reflects its activity. Time-lapse videomicroscopy and fluorescent recovery after photobleaching (FRAP) demonstrate that mobility of GFP-tagged nucleolar proteins is slower in the nucleolus than in the nucleoplasm. Fluorescent recovery rates change with inhibition of transcription, decreased temperature and depletion of ATP, indicating that recovery is correlated with cell activity. At the exit of mitosis, the nucleolar processing machinery is first concentrated in prenucleolar bodies (PNBs). The dynamics of the PNBs suggests a steady state favoring residence of processing factors that are then released in a control- and time-dependent manner. Time-lapse analysis of fluorescence resonance energy transfer demonstrates that processing complexes are formed in PNBs. Finally, the nucleolus appears at the center of several trafficking pathways in the nucleus.

Animals↗
PubMed2005

Tracking the interactions of rRNA processing proteins during nucleolar assembly in living cells.

Reorganization of the nuclear machinery after mitosis is a fundamental but poorly understood process. Here, we investigate the recruitment of the nucleolar processing proteins in the nucleolus of living cells at the time of nucleus formation. We question the role of the prenucleolar bodies (PNBs), during migration of the processing proteins from the chromosome periphery to sites of rDNA transcription. Surprisingly, early and late processing proteins pass through the same PNBs as demonstrated by rapid two-color four-dimensional imaging and quantification, whereas a different order of processing protein recruitment into nucleoli is supported by differential sorting. Protein interactions along the recruitment pathway were investigated using a promising time-lapse analysis of fluorescence resonance energy transfer. For the first time, it was possible to detect in living cells the interactions between proteins of the same rRNA processing machinery in nucleoli. Interestingly interactions between such proteins also occur in PNBs but not at the chromosome periphery. The dynamics of these interactions suggests that PNBs are preassembly platforms for rRNA processing complexes.

Anaphase↗
PubMed2005

The unusually large Plasmodium telomerase reverse-transcriptase localizes in a discrete compartment associated with the nucleolus.

Telomerase replicates chromosome ends, a function necessary for maintaining genome integrity. We have identified the gene that encodes the catalytic reverse transcriptase (RT) component of this enzyme in the malaria parasite Plasmodium falciparum (PfTERT) as well as the orthologous genes from two rodent and one simian malaria species. PfTERT is predicted to encode a basic protein that contains the major sequence motifs previously identified in known telomerase RTs (TERTs). At approximately 2500 amino acids, PfTERT is three times larger than other characterized TERTs. We observed remarkable sequence diversity between TERT proteins of different Plasmodial species, with conserved domains alternating with hypervariable regions. Immunofluorescence analysis revealed that PfTERT is expressed in asexual blood stage parasites that have begun DNA synthesis. Surprisingly, rather than at telomere clusters, PfTERT typically localizes into a discrete nuclear compartment. We further demonstrate that this compartment is associated with the nucleolus, hereby defined for the first time in P.falciparum.

Amino Acid Sequence↗
PubMed2004

Dynamics and compartmentation of the nucleolar processing machinery.

In active nucleoli, machineries involved in the biogenesis of ribosomal RNAs (rRNAs) are compartmentalized. The late rRNA processing proteins are localized in the granular component (GC). Here we investigate the behavior of these proteins when production of 28S is impaired and when this blockage is reversed. The 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) provokes dispersion of rDNA clusters and we demonstrate that DRB induces disconnection of the late rRNA processing proteins from the transcription sites. These processing proteins are still associated in independent masses without detectable 28S rRNA, indicating that compartmentation of the late rRNA processing machinery is not necessarily linked to processing activity. Removing DRB reverses this disconnection and promotes rRNA processing. Nucleolar reformation occurs in two successive steps, dynamic recruitment to transcription sites of the processing proteins, followed by rDNA compaction. We demonstrate that both steps are sensitive to temperature, suggesting an energy-dependent process. Traffic of processing proteins analyzed by fluorescence recovery after photobleaching is similar in masses disconnected from transcription sites and in the granular component of the active nucleolus. This suggests that protein dynamics and interactions, and not only their processing activity, determine compartmentation of the nucleolar machineries.

Active Transport, Cell Nucleus↗
PubMed2004

Aurora B -TACC1 protein complex in cytokinesis.

Taxins are a family of centrosomal proteins important for the regulation of mitosis and microtubule dynamics. Cytokinesis, the last step of M phase, is essential for chromosomal integrity and cell division. It is highly regulated and involves a reorganization of microtubules and actin filaments. We show here that TACC1 localizes diffusely to the midzone spindle in anaphase and strongly to the midbody during cytokinesis, indicating a possible involvement of this protein in the exit of M phase. TACC1 also relocalizes to the nucleolus in interphase. We demonstrate that TACC1 and the mitotic kinase Aurora B belong to the same complex during cytokinesis. We further show that Aurora B knocked down by RNA-mediated interference prevents the formation of the midbody - and consequently affects TACC1 localization at this site - and leads to abnormal cell division and multinucleated cells.

Anaphase↗
PubMed2004

[The nucleolus: structure, functions, amd associated diseases].

In eukaryotes, the nucleolus is the ribosome factory. The nucleolus is a very active large nuclear domain resulting from the equilibrium between level of ribosomal gene transcription, efficiency of rRNA processing and transport of the ribosomal subunits (40S and 60S) towards the cytoplasm. The ribosome production is regulated and is linked with cell growth and cell proliferation. The ribosome production is stopped during mitosis but the nucleolar machineries are inherited in daughter cells and the nucleolar reassembly is a very early event at the exit of mitosis. The nucleolus is also a multifunctional domain involved in nuclear architecture and specific interaction with some nuclear bodies. Finally, several human diseases appear to result from mutations of nucleolar proteins.

Cell Cycle↗
PubMed2003

Regulators of nucleolar functions.

The nucleolus is the ribosome factory and also a multifunctional domain that plays an important role in nuclear organization and function. Interestingly, ribosome biogenesis appears directly related to cell growth and proliferation. The nucleolus corresponds to a very dynamic nuclear domain resulting from an equilibrium between the level of ribosomal RNA synthesis and the efficiency of ribosomal RNA processing. Ribosome biogenesis is regulated throughout interphase and stops during mitosis. The mitotic silencing of transcription, the distribution of the processing machinery, as well as the link between transcription and processing were found to be dependent on cyclin-dependent kinase(s).

Animals↗
PubMed2002

Emerging concepts of nucleolar assembly.

The nucleolus is a large nuclear domain and the site of ribosome biogenesis. It is also at the parting of the ways of several cellular processes, including cell cycle progression, gene silencing, and ribonucleoprotein complex formation. Consequently, a functional nucleolus is crucial for cell survival. Recent investigations of nucleolar assembly during the cell cycle and during embryogenesis have provided an integrated view of the dynamics of this process. Moreover, they have generated new ideas about cell cycle control of nucleolar assembly, the dynamics of the delivery of the RNA processing machinery, the formation of prenucleolar bodies, the role of precursor ribosomal RNAs in stabilizing the nucleolar machinery and the fact that nucleolar assembly is completed by cooperative interactions between chromosome territories. This has opened a new area of research into the dynamics of nuclear organization and the integration of nuclear functions.

Animals↗
PubMed2002

Cyclin-dependent kinases govern formation and maintenance of the nucleolus.

In higher eukaryotic cells, the nucleolus is a nuclear compartment assembled at the beginning of interphase, maintained during interphase, and disorganized during mitosis. Even if its structural organization appears to be undissociable from its function in ribosome biogenesis, the mechanisms that govern the formation and maintenance of the nucleolus are not elucidated. To determine if cell cycle regulators are implicated, we investigated the putative role of the cyclin-dependent kinases (CDKs) on ribosome biogenesis and nucleolar organization. Inhibition of CDK1-cyclin B during mitosis leads to resumption of rDNA transcription, but is not sufficient to induce proper processing of the pre-rRNA and total relocalization of the processing machinery into rDNA transcription sites. Similarly, at the exit from mitosis, both translocation of the late processing machinery and pre-rRNA processing are impaired in a reversible manner by CDK inhibitors. Therefore, CDK activity seems indispensable for the building of functional nucleoli. Furthermore, inhibition of CDKs in interphasic cells also hampered proper pre-rRNA processing and induced a dramatic disorganization of the nucleolus. Thus, we propose that the mechanisms governing both formation and maintenance of functional nucleoli involve CDK activities and couple the cell cycle to ribosome biogenesis.

CDC2 Protein Kinase↗