Search

We have used polymerase chain reaction-mediated chemical mutagenesis (J.-J. Diaz, D. D. Rhoads, and D. J. Roufa, BioTechniques 11:204-211, 1991) to analyze the genetic fine structure of a human ribosomal protein gene, RPS14. Eighty-three DNA clones containing 158 random single-base substitution mutations were isolated. Mutant RPS14 alleles were tested for biological activity by transfection into cultured Chinese hamster cells. The resulting data permitted us to construct a map of the S14-coding sequence that is comparable to available fine-structure genetic maps of many prokaryotic and lower eukaryotic gene loci. As predicted from the multiplicity of protein-protein and protein-RNA interactions required for ribosomal protein transport and assembly into functional ribosomal subunits, the distribution of null mutations indicated that S14 is composed of multiple, functionally distinct polypeptide domains. Two of the protein's internal domains, designated domains B and D, were essential for S14 biological activity. In contrast, mutations which altered or deleted S14's amino-terminal 20 amino acid residues (domain A) had no observable effect on the protein's assembly and function in mammalian ribosomes. Interestingly, S14 structural domains deduced by in vitro mutagenesis correlate well with the RPS14 gene's exon boundaries.

International audience; Spi-1 and Fli-1 are ETS transcription factors recurrently deregulated in mouse erythroleukemia induced by Friend viruses. Since they share the same core DNA binding site, we investigated whether they may contribute to erythroleukemia by common mechanisms. Using inducible knockdown, we demonstrated that Fli-1 contributes to proliferation, survival, and differentiation arrest of erythroleukemic cells harboring an activated fli-1 locus. Similarly, we used inducible Fli-1 knockdown and either hexamethylenebisacetamide (HMBA)- or small interfering RNA-mediated Spi-1 knockdown to investigate their respective contributions in erythroleukemic cells harboring an activated spi-1 locus. In these cells, simple or double knockdown of both Spi-1 and Fli-1 additively contributed to induce proliferation arrest and differentiation. Transcriptome profiling revealed that virtually all transcripts affected by both Fli-1 knockdown and HMBA are affected in an additive manner. Among these additively downregulated transcripts, more than 20% encode proteins involved in ribosome biogenesis, and conserved ETS binding sites are present in their gene promoters. Through chromatin immunoprecipitation, we demonstrated the association of Spi-1 and Fli-1 on these promoters in Friend erythroleukemic cells. These data lead us to propose that the oncogenicity of Spi-1, Fli-1, and possibly other ETS transcription factors may involve their ability to stimulate ribosome biogenesis.

The third author's name was spelled incorrectly. The correct name is: Rouba Hage-Sleiman. The correct citation is: Beghin A, Belin S, Hage-Sleiman R, Brunet Manquat S, Goddard S, et al. (2009) ADP Ribosylation Factor Like 2 (Arl2) Regulates Breast Tumor Aggressivity in Immunodeficient Mice. PLoS ONE 4(10): e7478. doi:10.1371/journal.pone.0007478

US11 protein, one of herpes simplex virus type 1 (HSV-1) true late gene products, plays a role in the virally induced post-transcriptional control of gene expression. In addition, US11 expression also interferes with the cellular response to HSV-1 infection that can lead to apoptosis. We have previously shown that US11 expression enhanced the recovery of cellular protein synthesis and increased cell survival in response to thermal stress. Since heat shock can activate apoptosis, we tested for a possible anti-apoptotic behavior of US11. Here, we show that, in HeLa cells, US11 expression strongly reduced heat induced apoptosis, a phenomenon independent of Hsp expression and characterized by a delayed cytochrome c efflux from mitochondria and reduced caspase 3 activation. Moreover, US11 expression also protected against staurosporine induced apoptosis. Hence, our results favor an anti-apoptotic activity of US11 polypeptide that appears to be located at the level of mitochondria or upstream signaling pathways.

International audience; Proteomic analyses of human nucleoli provided molecular bases for an understanding of the multiple functions fulfilled by these nuclear domains. However, the biological roles of about 100 of the identified proteins are unpredictable. The present article describes the functional characterization of one of these proteins, ISG20L2. We demonstrate that ISG20L2 is a 3' to 5' exoribonuclease involved in ribosome biogenesis at the level of 5.8S rRNA maturation, more specifically in the processing of the 12S pre-rRNA. The use of truncated forms of ISG20L2 demonstrated that its N-terminal half promotes the nucleolar localization and suggested that its C-terminal half bears the exoribonuclease activity. Identification of the binding partners of ISG20L2 confirms its involvement in the biogenesis of the large ribosomal subunit. These results strongly support the notion that, in human, as it was demonstrated in yeast, 5.8S rRNA maturation requires several proteins in addition to the exosome complex. Furthermore, this observation sustains greatly the idea that the extremely conserved need for correctly processed rRNAs in vertebrates and yeast are achieved by close but different mechanisms.

A cDNA expression vector encoding Drosophila ribosomal protein S14 was transfected into cultured Chinese hamster ovary (CHO) cells that harbor a recessive RPS14 emetine resistance mutation. Transformants synthesized the insect mRNA and polypeptide and consequently displayed an emetine-sensitive phenotype. These observations indicate that the insect protein was accurately expressed and correctly assembled into functional mammalian 40S ribosomal subunits.

Herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) are members of the Herpesviridae family. HSV infections have been known since ancient times and are one of the most common communicable diseases in humans. Although infections are often subclinical, HSV can cause mild to severe diseases, especially in immunocompromised patients. Herpes simplex viruses establish latency in the nuclei of neuronal cells and may reactivate, with or without symptoms, throughout the host's lifetime. Over one third of the world's population suffer from recurrent HSV infections several times a year and are thus capable of transmitting HSV by close personal contact. There are few drugs licensed for the treatment of HSV infections. Most target the viral DNA polymerase, and indeed acyclovir remains the reference treatment some thirty years after its discovery! Extensive clinical use of this drug has led to the emergence of resistant viral strains, mainly in immunocompromised patients. This highlights the crucial need for the development of new anti-herpes drugs that can inhibit infection by both wild-type viruses and drug-resistant strains. Over the last few years, significant efforts have been made to set up a range of strategies for the identification of potential new anti-viral drugs. One alternative is to develop drugs with different mechanisms of action. The present article reviews potential viral and cellular targets that are now known to be involved in HSV infection and for which specific inhibitors with anti-HSV activity, at least in cell culture, have been identified.

All the available antiherpetic drugs are directed against viral proteins. Their extensive clinical use has led to the emergence of resistant viral strains. There is a need for the treatment of herpes infections due to resistant strains, especially for immunocompromised patients. To design new kinds of drugs, we have developed a strategy to identify cellular targets. Herpes simplex virus type 1 (HSV-1) infection is concomitant to a repression of most host protein synthesis. However, some cellular proteins continue to be efficiently synthesized. We speculated that some of them could determine the outcome of infection. Since two polyamines, spermidine and spermine, are components of the HSV-1 virions, we investigated whether enzymes involved in their synthesis could be required for viral infection. We show that inhibition of S-adenosyl methionine decarboxylase, a key enzyme of the polyamine metabolic pathway, prevents HSV-1 infection. Inhibition of polyamine synthesis prevents infection of culture cells with HSV-1 laboratory strains as well as clinical isolates that are resistant to the conventional antiviral drugs acyclovir and foscarnet. Our data provide the opportunity to develop molecules with a novel mechanism of action for the treatment of herpes infection.

Homeodomain-interacting protein kinase 2 (HIPK2) is a nuclear serine/threonine kinase of the subfamily of dual-specificity Yak1-related kinase proteins. HIPK2 was first described as a homeodomain-interacting protein kinase acting as a corepressor for homeodomain transcription factors. More recently, it was reported that HIPK2 plays a role in p53-mediated cellular apoptosis and could also participate in the regulation of the cell cycle. US11 protein of herpes simplex virus type 1 is a multifunctional protein involved in the regulation of several processes related to the survival of cells submitted to environmental stresses by mechanisms that are not fully elucidated. In an attempt to better understand the multiple functions of US11, we identified cellular binding partners of this protein by using the yeast two-hybrid system. We report that US11 interacts with HIPK2 through the PEST domain of HIPK2 and that this interaction occurs also in human cells. This interaction modifies the subcellular distribution of HIPK2 and protects the cell against the HIPK2-induced cell growth arrest.

Polyamines are involved in the control of the cell cycle and cell growth. In murine kidney, testosterone enhances gene expression of ornithine decarboxylase (ODC), the first enzyme in polyamine biosynthesis. In this study, we document the time course effect of testosterone on 1) gene expression of ODC, antizyme 1 (AZ1), and spermidine/spermine- N1-acetyltransferase ( N1-SSAT); 2) ODC activity in proximal convoluted tubules (PCT) and cortical proximal straight tubules (CPST); and 3) renal polyamine levels. Female mice were treated with testosterone for a period of 1, 2, 3, and 5 consecutive days. ODC gene expression was extremely low in kidneys of untreated female mice compared with that of males. Consequently, the renal putrescine level was sevenfold lower in females than in males, whereas spermidine and spermine levels did not differ between sexes. In female kidneys, testosterone treatment sharply increased ODC mRNA and protein levels as well as ODC activity. Testosterone increased the expression of ODC in PCT and CPST over different time courses, which suggests that ODC activity is differentially regulated in distinct tubules. The expression of AZ1 and N1-SSAT mRNA was similar in male and female mouse kidneys. Testosterone treatment enhanced AZ1 and N1-SSAT mRNA levels in a time-dependent manner by unknown molecular mechanisms. Putrescine and spermidine levels increased after testosterone treatment in female kidneys. Surprisingly, although ODC protein and activity were undetectable in female kidneys, the levels of AZ1 mRNA and protein were similar to those in males. Therefore, one may propose that ODC protein could be continuously degraded by AZ1 in female kidneys.

The notion of a “plurifunctional” nucleolus is now well established. However, molecular mechanisms underlying the biological processes occurring within this nuclear domain remain only partially understood. As a first step in elucidating these mechanisms we have carried out a proteomic analysis to draw up a list of proteins present within nucleoli of HeLa cells. This analysis allowed the identification of 213 different nucleolar proteins. This catalog complements that of the 271 proteins obtained recently by others, giving a total of ∼350 different nucleolar proteins. Functional classification of these proteins allowed outlining several biological processes taking place within nucleoli. Bioinformatic analyses permitted the assignment of hypothetical functions for 43 proteins for which no functional information is available. Notably, a role in ribosome biogenesis was proposed for 31 proteins. More generally, this functional classification reinforces the plurifunctional nature of nucleoli and provides convincing evidence that nucleoli may play a central role in the control of gene expression. Finally, this analysis supports the recent demonstration of a coupling of transcription and translation in higher eukaryotes.

At present, the effect of herpes simplex virus infection on the entire proteomes of infected cells is very poorly documented. Following several studies performed over the past few years, the modifications of a sub-cellular fraction induced by herpes simplex virus type 1 can be documented. These studies were performed in order to characterize the virally-induced modifications of a major component of the translational apparatus, the ribosomes. The very basic nature of most of the ribosomal proteins renders them very difficult to separate using isoelectric focusing (IEF). Therefore these studies were achieved using several different but related two-dimensional electrophoretic systems which allowed several two-dimensional ribosomal protein maps to be built. Comparison of the ribosomal protein maps built from non-infected cells with those built from infected cells demonstrated that infection by herpes simplex virus type 1 (HSV-1) induces important modifications of ribosomes: (i) non-reversible phosphorylation of ribosomal protein S6; (ii) unusual phosphorylation of several proteins of the small and the large subunits; and (iii) association of viral and cellular proteins to the ribosomal fraction. An overview of these published studies is presented in this review.

Genome sequencing projects produce large amounts of information that could be translated into potential protein sequences. Such amounts of material continuously increase protein database sizes. At present, 22 times more protein sequences are available in the SWISS-PROT and TrEMBL databases than 8 years ago in SWISS-PROT. One of the methods of choice for protein identification makes use of specific endoproteolytic cleavage followed by matrix-assisted laser desorption/ionisation mass spectrometric (MALDI-MS) analysis of the digested product. Since 1993, when this technique was first demonstrated, the conditions required for a correct identification have changed dramatically. Whilst 4-5 peptides with an uncertainty of 2-3 Da were sufficient for a correct identification in 1993, 10-13 peptides with less than 60 ppm mass error are now required for human and E. coli proteins. This evolution is directly related to the continuous increase in protein database sizes, which causes an increase in the number of false positive matches in identification results. Use of an information complement deduced from the primary protein sequence, in the process of identification by peptide mass fingerprints, can help to increase confidence in the identification results. In this article, we propose the exchange of labile hydrogen atoms with deuterium atoms to provide an alternative information complement. The exchange reaction with optimised techniques has shown an average 95% of hydrogen/deuterium (H/D) exchange on tryptic peptides. This level of exchange was sufficient to single out one or more peptides from a list of potential candidate proteins due to the dependence of H/D exchange on the peptide primary structure. This technique also has clear advantages in the identification of small proteins where direct protein identification is impaired by the limited number of endoproteolytic peptides. Then, information related to primary sequence obtained with this technique could help to identify proteins with high confidence without any expensive tandem mass spectrometry instruments.

Herpes simplex virus type 1 (HSV-1) gene expression is concomitant with a selective shutoff of host protein synthesis. While the synthesis of the vast majority of cellular proteins is inhibited immediately after infection, several cellular proteins continue to be synthesized, even during the late phase of infection. Because these cellular proteins may intervene in the life cycle of the virus, we undertook two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analyses to evaluate the proportion of cellular proteins that is represented by these particular proteins. Human cells were infected with HSV-1. At different times after infection, proteins were labeled with 35S just prior to harvesting. The rate of synthesis of a set of 183 acidic host proteins, as well as that of ribosomal proteins, was measured during the course of infection, after separation by 2-D PAGE. As expected, HSV-1 induces a strong inhibition of host protein synthesis immediately after infection. However, the synthesis of basic ribosomal proteins and that of an unexpected high proportion of the sub-set of cellular proteins analyzed is sustained or stimulated during HSV-1 infection. A 2-D PAGE analysis outlining the expression patterns of these proteins at different times of infection is presented.

The fate of ornithine in the nephron of the female OF-1 Swiss mouse remains unknown. The aim of the present study was to identify the nephron segments containing the key enzymes involved in ornithine metabolism: ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT). Viable tubules isolated by microdissection were incubated with [1-14C]ornithine to study the oxidative pathway. Other tubules were permeabilized to measure the ODC activity. Ornithine was decarboxylated in all intact tubules. Gabaculine, a suicide inhibitor of OAT, and rotenone sharply decreased the production of 14CO2 from [1-14C]ornithine. No ODC activity was found in permeabilized tubules isolated from untreated mice. Testosterone increased ODC activity in the proximal tubule substantially and to a minor extent in other nephron segments. In situ hybridization showed ODC messenger ribonucleic acid (mRNA) to be absent in kidneys of untreated females but abundant in the cortex and the outer stripe of the outer medulla of testosterone-treated female mice. The whole proximal tubule contained a great density of silver grains corresponding to ODC mRNA. In conclusion, no basal ODC activity was found in the nephron of female mice. The testosterone-inducible ODC is localized mainly in the proximal tubule, but is also present in distal tubules and collecting ducts. OAT is distributed along the whole nephron, but its activity is higher in proximal tubules than in distal tubules.

Herpes simplex virus type 1 (HSV- 1) US11 protein is an RNA-binding protein which is able to mediate post-transcriptional transactivation of human T-lymphotropic virus type I (HTLV-I) envelope glycoprotein gene expression by interacting with the Rex responsive element (XRE) located at the 3' end of the env mRNA. In view of this functional activity, and because US11 protein is capable of substituting for HTLV-I Rex protein, it was hypothesized that US11 protein should exhibit at least two functional domains, an RNA-binding domain for specific interaction with the target RNA, and an effector domain involved in transport and translation of this mRNA. Recombinant US11 wild-type and deleted proteins were tested for their ability (i) to bind to the XRE and to HSV-1 UL34 RNA, the natural target of US11 protein, and (ii) to transactivate HTLV-I env gene expression. The C-terminal half of US11 protein, consisting of 20-24 XPR repeats, was necessary and sufficient to mediate RNA-binding with a high affinity and specificity. Structure prediction analyses showed the likely conformation of this domain to be that of a polyproline type II helix. Localized within the first 40 amino acids of the N-terminal region of US11 protein was the effector domain, deletion of which created US11(delta1-40), a trans-dominant negative mutant. These results demonstrate structural differences between US11 protein and proteins like Rex and Rev, despite their functional similarities.

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.

One of the herpes simplex virus type 1 (HSV-1) true late gene products, Us11 protein, is brought into the cell by the infecting virion and may play a role in the virally-induced post-transcriptional control of gene expression. Us11 protein forms large oligomers, exhibits RNA binding features, concentrates into the nucleolus and is able to replace Rex protein in post-transcriptional control of human T-cell leukemia/lymphoma virus type I (HTLV-I) expression. As heat shock drastically alters protein synthesis, and because HSV-1 infection stimulates heat shock protein (Hsp) expression, we analyzed the consequence of heat shock in HeLa cells expressing Us11 alone, either transiently or constitutively. No detectable modification of the overall pattern of protein synthesis was observed in cells growing at normal temperatures, including no induction of Hsp expression or accumulation. However, Us11 protein expression induced an enhanced recovery of protein synthesis after heat shock. Moreover, the level of Us11 protein-mediated protection of protein synthesis was similar to that observed for cells made thermotolerant, but only when submitted to a mild heat shock. Finally, Us11 protein expression induced in cells an enhanced survival to heat shock.

Because synthesis of rRNA persists late during herpes simplex type 1 infection and because S6 phosphorylation is always correlated with efficient translation of ribosomal protein mRNA, we tested the hypothesis that ribosomal protein synthesis and ribosome biogenesis could persist after infection. At different times after infection, proteins were labelled with 35S for 1 h before harvesting and ribosomes were purified. Measurement of radioactivity incorporated into individual ribosomal proteins separated by two-dimensional PAGE demonstrated that ribosomal proteins are still synthesized and assembled into mature ribosomes up to late times during infection, while synthesis of beta-actin is severely inhibited. During expression of late genes, ribosome biogenesis was estimated to be 58% of that of the control as judged by [3H]uridine incorporation into rRNA. As for beta-actin mRNA, the level of ribosomal protein mRNA decreased progressively from the beginning of infection, reaching about 30% of the control level during expression of late genes. Taken together, these results demonstrate that ribosomal proteins are still synthesized up to the late time of infection and efficiently assembled into mature ribosomes, while there is a severe shutoff of the synthesis of other cellular proteins.

The amino acid sequence of the rat 60S ribosomal subunit protein L10 was deduced from the sequence of nucleotides in two recombinant cDNAs and confirmed by determination of the NH2-terminal amino acid sequence in the protein. Ribosomal protein L10 has 213 amino acids (the NH2-terminal methionine is removed after translation of the mRNA); the molecular weight is 24,456. Hybridization of the cDNA to digests of nuclear DNA suggests that there are 8 to 10 copies of the L10 gene. The mRNA for the protein is about 900 nucleotides in length. Rat L10 is related to ribosomal proteins from other eukaryotes. Ribosomal protein L10 is, in addition, the mammalian homolog of the chicken Jun-binding protein and is nearly identical to a putative Wilms' tumor suppressor. This is a presumptive example, of which there are many others, of an extraribosomal function of a ribosomal protein.

The distribution of Us11 RNA and of its encoded protein have been investigated at the ultrastructural level in HeLa cells transiently expressing the Us11 gene of herpes simplex virus type 1. In these transfected cells, Us11 protein accumulates at sites identical to those of lytically infected cells, i.e., in nucleoli and in regions of the cytoplasm that contain ribosomes. Us11 RNA and polyadenylated RNA are scattered over the ribosome-rich areas of the cytoplasm. They also accumulate in the nucleoplasm on clustered ribonucleoprotein (RNP) fibrils but also in clusters of interchromatin granules, some of them contiguous to nucleoli. However they are never found in nucleoli. These data reveal the involvement of interchromatin granules in some steps of Us11 mRNA maturation and/or transport.

The Us11 protein is a true late gene product of herpes simplex virus type 1 (HSV-1), whose exact function is unknown but which exhibits RNA-binding properties and which is phosphorylated on serine residues. In order to determine whether the Us11 protein is phosphorylated by cellular kinase(s) or by virally encoded kinase(s), the Us11 gene has been cloned and transiently expressed in HeLa cells. In addition, HeLa-derived cell lines have been selected for their ability to express Us11 protein constitutively. 32P-Labeling and analysis by two-dimensional electrophoresis of transiently and constitutively expressed Us11 protein demonstrated that, indeed, multiple phosphorylation of the protein occurs in absence of HSV-1 genome expression, indicating that the protein behaves as a natural substrate for cellular kinase(s). In addition, a sequence heterogeneity of the Us11 protein, due to a difference in the number of SPREPR repeats, has been characterized between different strains of HSV-1.

In addition to an irreversible stimulation of S6 ribosomal protein phosphorylation, there is a modification of a subset of ribosomal proteins by phosphorylation after herpes simplex virus type 1 (HSV-1) infection. Moreover, in the course of this infection, three additional phosphorylated proteins can be extracted from ribosomes and separated by two-dimensional electrophoresis (2-DE) of total ribosomal proteins. One of them exhibits an identical molecular mass to L30, while being more acidic. This protein is phosphorylated on serine residues. The kinetics of appearance of this protein in the ribosomal fraction correlated with a decrease in L30 staining, as shown by 2-DE. Determination of the N-terminal amino acid sequence of this extra phosphoprotein and of L30-derived peptides demonstrated the identity of these two proteins.

The sequence coding for the 5′ untranslated region (UTR) of ICP22 mRNA of herpes simplex virus type 1 has been tested for its ability to regulate gene expression. This sequence was placed in frame with the chloramphenicol acetyltransferase (CAT) coding sequence and under the control of the simian virus 40 early promoter-enhancer. Under these conditions, the sequence coding for the 5′UTR led to an increase of about 13-fold in CAT activity, measured during transient expression. The use of mutants with progressive deletions within the sequence coding for the 5′UTR allowed localization of the sequence responsible for the enhancement of gene expression to the first exon of the ICP22 gene. Precise quantification of hybrid ICP22-CAT mRNA showed that the sequence coding for the 5′UTR induced an increase in the amounts of transcripts, which resulted in a parallel increase in CAT activity. This increase in the level of hybrid ICP22-CAT mRNA is not the result of an increase in mRNA stability, nor is it due to more efficient nucleo-cytoplasmic transport of the transcripts. Moreover, the distribution of hybrid mRNA in the different ribosomal populations indicates that the 5′UTR of ICP22 mRNA does not induce a preferential recruitment of the transcripts by the translational apparatus. Taken together, these results indicate that a cis-acting element located in the sequence coding for the 5′UTR of ICP22 mRNA can mediate a high level of gene expression independently of the viral promoter and of viral trans-acting factors.

Adrenocorticotropic hormone (ACTH) and melanocortin peptides (alpha, beta and gamma MSH) have numerous activities in both central nervous system and peripheral tissues, namely the adrenals. Recently, five melanocortin receptors were cloned and characterized. We report here the cloning, pharmacological characterization and expression of the rat fifth melanocortin receptor (MC5), starting from the dopamine D3 receptor sequence to screen a genomic DNA library. The MC5 comprises a sequence of 325 amino acids, displaying 45-62% identity with other melanocortin receptors and 82% identity with its human counterpart that we cloned thereafter. The sequence of the latter is identical to that of a so-called 'MC2' receptor (Chhajlani et al., 1993, Biochem. Biophys. Res. Comm. 195, 866-873). The MC5, stably expressed in CHO cells, mediates increase in cAMP accumulation with a characteristic pharmacology: alpha MSH is twice as potent as NDP alpha MSH, 10 times as ACTH and 100 times as gamma MSH. Very low expression levels were detected in brain, while high levels were found in adrenals, stomach, lung and spleen. In addition, in situ hybridization studies show the MC5 expressed in the three layers of the adrenal cortex, predominantly in the aldosterone-producing zona glomerulosa cells.

International audience; In contrast with many physiological studies suggesting that histamine H2 receptors are present on acid-secreting parietal cells of the gastric epithelium, it was recently shown that immune cells in the lamina propria are the only cells expressing H2-receptor mRNAs (Mezey and Palkovits, Science, 1992, 258, 1662-1665). We have reinvestigated the cellular localization of H2 receptors in the rat stomach by visualizing both the H2 receptor mRNA and the H2-receptor protein itself. In situ hybridization histochemistry performed with an antisense riboprobe for the rat H2 receptor, and autoradiographic distribution of 125I-aminopotentidine binding sites, a highly selective H2-receptor ligand, did not show any labeling of the lamina propria. Signals were clearly and solely detected in the gastric epithelium, the strongest being observed in the upper part of the glands where the H2 receptor gene transcripts were only detected within parietal cells. In situ hybridization performed with an antisense riboprobe for L-histidine decarboxylase mRNA confirmed the basal localization of the histamine-synthetizing cells in the rat gastric gland, at some distance from parietal histamine-sensitive cells.

Our knowledge of dopamine receptor diversity has markedly increased during the past few years as a result of discovery of five distinct genes, splice variants and polymorphic receptors. The genes can be classified in two subfamilies: the intronless genes that encode the D1 and D5 receptors positively linked to adenylyl cyclase and genes with introns that encode the two isoforms of the D2 receptor and the D3 and D4 receptors. The various dopamine receptor subtypes can be distinguished by their sequence, intracellular signalling systems, pharmacology and localisation. The localisation of the D3 receptor in the shell of nucleus accumbens suggests its participation in brain reward circuits and actions of substances of abuse.

Microsequencing of a cyanogen bromide peptide obtained from a basic phosphoprotein co-sedimenting with purified ribosomes extracted from herpes simplex virus type 1-infected human epidermoid carcinoma 2 cells identified this protein as a product of the true late US11 gene. An antibody was raised against a recombinant fusion protein expressed in Escherichia coli from a plasmid carrying 75% of the US11 coding sequence including the carboxy terminus. This antibody was used to prode Western blots carried out under various conditions of one- and two-dimensional electrophoresis. The electrophoretic behaviour of the immunoreactive proteins offered further proof that they were indeed products of the US11 gene. This US11 protein, which has phosphates on multiple serine residues, is brought into the cell by the virion and found to be present within ribosome fractions early after infection. This association with ribosomes is non-specific and due to probable aggregation or oligomerization of this proline-rich basic protein allowing its co-sedimentation with ribosomes during the different subcellular fractionation steps used for the purification of ribosomal subunits.

We describe a novel approach for the isolation of null mutations in a vital Chinese hamster ovary (CHO) cell housekeeping gene. Our experimental strategy required introduction of an expressible DNA clone encoding a recessive emetine-resistance allele of ribosomal protein S14 into wild-type CHO cells. Transgene heterozygote (TGH) cell lines, which harbor multiple emetine-resistance S14 transgenes, survive mutations that inactivate the CHO RPS14 locus by virtue of the transgenes' biological function. Null mutations in RPS14 yield TGH clones that display the transgene's drug-resistance phenotype. A large collection of emetine-resistant clones was isolated from one TGH cell line and shown to consist of three types of S14 mutations: (1) nonsense null mutations in the RPS14 protein coding sequence; (2) missense null mutations that affect S14 amino acid residues that have been conserved stringently during eukaryotic evolution; and (3) a recurrent missense mutation that results in a new, functional RPS14 emetine-resistance allele.

Influenza A are nuclear replicating viruses which hijack host machineries in order to achieve optimal infection. Numerous functional virus–host interactions have now been characterized, but little information has been gathered concerning their link to the virally induced remodeling of the host cellular architecture. In this study, we infected cells with several human and avian influenza viruses and we have analyzed their ultrastructural modifications by using electron and confocal microscopy. We discovered that infections lead to a major and systematic disruption of nucleoli and the formation of a large number of diverse viral structures showing specificity that depended on the subtype origin and genomic composition of viruses. We identified NS1 and M1 proteins as the main actors in the remodeling of the host ultra-structure and our results suggest that each influenza A virus strain could be associated with a specific cellular fingerprint, possibly correlated to the functional properties of their viral components.

Avian erythroblastosis virus (AEV-ES4), a transforming avian retrovirus, transforms chicken embryo fibroblasts (CEFs) in culture and induces the maintenance of ribosomal protein S6 phosphorylation in the absence of serum. This effect is less pronounced after AEV-ES4 transformation than after transformation by Rous sarcoma virus (PR-RSV A). However, our results indicate that the two viruses induce an activation of the same S6 phosphokinase, as evidenced by the identity of S6 phosphopeptides and phosphoaminoacids in the two cases. Moreover this activation is performed through a protein kinase C-independent pathway. Expression of the v-erbA oncogene alone, which enhances the growth potential of CEFs, is not able to maintain S6 phosphorylation either in the absence of serum or in the presence of low serum concentration (0.5%). Expression of the v-erbB oncogene alone is responsible for all these AEV-ES4-induced effects. Furthermore, the maintenance of S6 phosphorylation in the absence of serum might be correlated with the degree of transformation of AEV-ES4-infected CEFs. These results show that S6 phosphorylation is one of the biochemical mechanisms deregulated by v-erbB expression and is involved in the transformation process.Avian erythroblastosis virus (AEV-ES4), a transforming avian retrovirus, transforms chicken embryo fibroblasts (CEFs) in culture and induces the maintenance of ribosomal protein S6 phosphorylation in the absence of serum. This effect is less pronounced after AEV-ES4 transformation than after transformation by Rous sarcoma virus (PR-RSV A). However, our results indicate that the two viruses induce an activation of the same S6 phosphokinase, as evidenced by the identity of S6 phosphopeptides and phosphoaminoacids in the two cases. Moreover this activation is performed through a protein kinase C-independent pathway. Expression of the v-erbA oncogene alone, which enhances the growth potential of CEFs, is not able to maintain S6 phosphorylation either in the absence of serum or in the presence of low serum concentration (0.5%). Expression of the v-erbB oncogene alone is responsible for all these AEV-ES4-induced effects. Furthermore, the maintenance of S6 phosphorylation in the absence of serum might be correlated with the degree of transformation of AEV-ES4-infected CEFs. These results show that S6 phosphorylation is one of the biochemical mechanisms deregulated by v-erbB expression and is involved in the transformation process.

We describe an efficient, PCR-mediated protocol for random chemical mutagenesis of cloned duplex DNAs. The method involves a single molecular cloning step and is compatible with a wide variety of recombinant DNA vectors. To illustrate the procedure, we report the nitrous acid mutagenesis of a human ribosomal protein S14 cDNA fragment.

Nuclear distribution and migration of herpes simplex virus type 1 Us11 transcripts were studied in transient expression at the ultrastructural level and compared to that of RNA polymerase II protein. Transcription was monitored by autoradiography following a short pulse with tritiated uridine. Us11 transcripts accumulated mainly over the foci of intermingled RNP fibrils as demonstrated by the presence of silver grains localizing incorporated radioactive uridine superimposed to these structures in which the presence of Us11 RNA and poly(A) tails was previously demonstrated. Silver grains were also scattered over the remaining nucleoplasm but not in the clusters of interchromatin granules, and over the dense fibrillar component of the nucleolus as in control, nontransfected HeLa cells. Pulse-chase experiments revealed the transient presence of migrating RNA in the clusters of interchromatin granules. RNA polymerase II was revealed by immunogold labeling following the use of two monoclonal antibodies: mAb H5, which recognizes the hyperphosphorylated form of the carboxy-terminal domain (CTD) of the molecule, and mAb 7C2, which recognizes both its hyperphosphorylated and unphosphorylated forms. The two mAbs bind to the newly formed Us11 transcription factories and the clusters of interchromatin granules of transfected cells. In control cells, however, clusters of interchromatin granules were labeled with mAb H5 but not with mAB 7C2. Taken together, our data demonstrate the involvement of the clusters of interchromatin granules in the intranuclear migration of Us11 RNA in transient expression. They also suggest the occurrence of changes in the accessibility of the RNA polymerase II CTD upon expression of the Us11 gene after transfection by exposing some epitopes, otherwise masked in nontransfected cells.

Nuclear distribution and migration of herpes simplex virus type 1 Us11 transcripts were studied in transient expression at the ultrastructural level and compared to that of RNA polymerase II protein. Transcription was monitored by autoradiography following a short pulse with tritiated uridine. Us11 transcripts accumulated mainly over the foci of intermingled RNP fibrils as demonstrated by the presence of silver grains localizing incorporated radioactive uridine superimposed to these structures in which the presence of Us11 RNA and poly(A) tails was previously demonstrated. Silver grains were also scattered over the remaining nucleoplasm but not in the clusters of interchromatin granules, and over the dense fibrillar component of the nucleolus as in control, nontransfected HeLa cells. Pulse-chase experiments revealed the transient presence of migrating RNA in the clusters of interchromatin granules. RNA polymerase II was revealed by immunogold labeling following the use of two monoclonal antibodies: mAb H5, which recognizes the hyperphosphorylated form of the carboxy-terminal domain (CTD) of the molecule, and mAb 7C2, which recognizes both its hyperphosphorylated and unphosphorylated forms. The two mAbs bind to the newly formed Us11 transcription factories and the clusters of interchromatin granules of transfected cells. In control cells, however, clusters of interchromatin granules were labeled with mAb H5 but not with mAB 7C2. Taken together, our data demonstrate the involvement of the clusters of interchromatin granules in the intranuclear migration of Us11 RNA in transient expression. They also suggest the occurrence of changes in the accessibility of the RNA polymerase II CTD upon expression of the Us11 gene after transfection by exposing some epitopes, otherwise masked in nontransfected cells.

Breast cancer is classified into several subtypes with varying clinical features. In particular, the so-called triple negative subtype is associated with a poor prognosis and a higher probability of recurrence. This is mainly due to the lack of specific targeted therapies. It is then necessary to identify new therapeutic targets in order to develop new drugs. Ribosome biogenesis is altered in most cancers due to the deregulation of the main oncogenic and tumor suppressor pathways, which regulate the activity of RNA Polymerase I, the enzyme responsible for ribosomal RNA transcription. Inhibition of ribosome biogenesis has been validated as a therapeutic tool, on the basis of three compounds that inhibit RNA Polymerase I activity (CX-5461, CX-3543 and BMH-21) that target cancer cells versus healthy cells. Their anti-cancer activity has been reported in several cancers, but the potential benefit of targeting ribosome biogenesis in certain types of breast cancer, including triple negative breast cancer, has not been evaluated yet. Although very little is known about ribosome biogenesis in triple negative breast cancer, there is now urgent need for new therapeutic tools and the potential of inhibiting ribosome biogenesis deserves to be evaluated. In addition, ribosome biogenesis depends on many maturation factors that represent a new family of targets to be explored. Here, the targeting of Fibrillarin is proposed. This may be relevant, as fibrillarin is often overexpressed in triple negative breast cancer and is associated with poor prognosis and risk of recurrence. Several cytotoxic mechanisms have been described in various cancer models upon treatment with RNA Pol I inhibitors and the elicited response appears to be model-specific. At present, since the effect on cells is unpredictable, the characterization of such a response in study models is not unfounded, especially since triple negative breast cancer is overly heterogeneous. The two main objectives of this thesis are: 1. To investigate the sensitivity of triple negative breast cancer lines to inhibition of ribosome biogenesis by pharmacological treatment targeting RNA Polymerase I or by genetic silencing of Fibrillarin; 2. To determine the mechanisms inducing the cytotoxic effect of inhibition of ribosome biogenesis in this model. In this work, using a large panel of cancer cell lines representing four different subtypes of triple negative breast cancer, it is shown that inhibition of ribosome biogenesis by BMH-21 and CX-5461 compounds induces strong cell growth inhibition and loss of the tumorigenic phenotype in vitro. In accordance with the heterogeneity of the model, triple negative cell lines have a wide range of sensitivity, but without association with subtypes according to common classification systems. Regarding induced cellular responses, an interesting phenomenon was observed: although BMH-21 causes apoptosis, no evidence of apoptosis was observed during treatment with CX-5461. In addition, it is demonstrated that inhibition of ribosome biogenesis downstream of RNA Polymerase I, by targeting Fibrillarin, also induces strong inhibition of cell growth, associated with an apoptotic response. Overall, these results suggest that inhibition of ribosome biogenesis could be a promising therapeutic strategy for triple negative breast cancer and validate Fibrillarin and ribosome RNA maturation as a new attractive therapeutic target, Le cancer du sein est classé en plusieurs sous-types dont les caractéristiques cliniques varient considérablement. En particulier, le sous-type dit triple-négatif est associé à un mauvais pronostic et à une probabilité plus élevée de récidive. Cela s'explique principalement par l'absence de thérapies ciblées spécifiques. Il est nécessaire d'identifier de nouvelles cibles thérapeutiques, afin de développer de nouveaux médicaments. La biogénèse des ribosomes est altérée dans la plupart des cancers, en raison de la dérégulation des principales voies oncogèniques et suppresseurs de tumeur, qui régulent l'activité de la ARN Polymerase I, l'enzyme responsable de la transcription de l'ARN ribosomique. L'inhibition de la biogénèse des ribosomes a été validée comme outil thérapeutique, à partir de trois composés qui inhibent l'activité de l'ARN Polymerase I (CX-5461, CX-3543 et BMH-21) qui ciblent les cellules cancéreuses par rapport aux cellules saines. Leur activité anti-tumorale a été rapportée dans plusieurs types de cancer, mais l'avantage potentiel de cibler la biogenèse des ribosomes dans certains types de cancer du sein, notamment les triple-négatifs, n'a pas été étudié à ce jour. Bien que très peu de choses soient connues sur la biogénèse des ribosomes dans le cancers du sein triple-négatifs, il y a aujourd'hui un besoin urgent de nouveaux outils thérapeutiques et le potentiel de l'inhibition de la biogenèse des ribosomes doit être évalué. Par ailleurs, la biogenèse des ribosomes dépend de nombreux facteurs de maturation qui représentent une nouvelle famille de cibles à explorer. Ici, le ciblage de la Fibrillarine est proposé. Cela pourrait être pertinent, car la fibrillarine est souvent surexprimée dans les cancers du sein triple-négatifs et est associée à un mauvais pronostic et à un risque de récidive. Plusieurs mécanismes cytotoxiques ont été décrits dans divers modèles de cancer lors du traitement par des inhibiteurs de l'ARN Pol I et la réponse obtenue semble être propre à chaque modèle. À l'heure actuelle, comme l'effet sur les cellules est imprévisible, la caractérisation d'une telle réponse dans les modèles d'études n'est pas sans fondement, d'autant plus que les cancer du sein triple-négatifs sont très hétérogènes. Les deux principaux objectifs de cette thèse sont donc : 1. Étudier la sensibilité de lignées de cancer du sein triple négatif à l'inhibition de la biogénèse des ribosomes par un traitement pharmacologique ciblant l'ARN Polymerase I ou bien par ciblage génétique de la Fibrillarine ; 2. Déterminer les mécanismes induisant l'effet cytotoxique de l'inhibition de la biogenèse des ribosomes dans ce modèle d'étude. Dans ce travail, en utilisant un large panel de lignées de cellules cancéreuses représentant quatre sous-types différents des triple-négatifs, il est montré que l'inhibition de la biogénèse des ribosomes par les composés BMH-21 et CX-5461 induit une forte inhibition de la croissance cellulaire et une perte du phénotype tumorigène in vitro. Conformément à l'hétérogéneité du modèle, les lignées cellulaires triple-négatives présentent une large gamme de sensibilité, mais sans association avec les sous-types selon les systèmes de classification courants. Concernant les réponses cellulaires induites, un phénomène intéressant a été observé : bien que le BMH-21 provoque l'apoptose, aucun signe d'apoptose n'a été observé lors du traitement par CX-5461. De plus, les résultats montrent que l'inhibition de la biogénèse des ribosomes en aval de l'ARN Polymerase I, par ciblage génique de la Fibrillarine, induit également une forte inhibition de la croissance cellulaire, associée à une réponse apoptotique. Dans l'ensemble, ces résultats suggèrent que l'inhibition de la biogénèse des ribosomes pourrait constituer une stratégie thérapeutique prometteuse pour le cancer du sein triple-négatif et positionnent la Fibrillarine et la maturation des ARN ribosomiques comme de nouvelles cibles thérapeutiques intéressante

Ribosomes are responsible of translating mRNAs to proteins. Alterations of ribosome composition modify its translation activity and favour tumourigenesis. Identification of ribosomes composition alterations in breast cancers might correspond to a new mechanism responsible of mammary tumourigenesis and might open up novel therapeutic approaches. Indeed breast cancers represent the first cause of women mortality due to cancers and their heterogeneity induces an important therapeutic problem.In this context, alterations of ribosomes composition were determined in human cohorts and in EMT (Epithelial to Mesenchymal Transition) cellular models, the EMT being a process involved in mammary tumourigenesis. This studies identify : (i) two factors involved in ribosome biogenesis, FBL (fibrillarin) and NCL (nucleolin) whose expression variations are associated with poor prognosis in patients and (ii) variations of ribosome composition and its translational activity in EMT. Altogether, this data support the presence of ribosomes composition alterations in breast cancers; Les ribosomes sont responsables de la traduction des ARNm en protéines. Des modifications de la composition des ribosomes altèrent son activité de traduction et favorisent la tumorigenèse. L’identification des altérations de composition des ribosomes dans les cancers du sein pourrait être un nouveau mécanisme de tumorigenèse mammaire et ouvrir de nouvelles perspectives thérapeutiques. En effet, les cancers du sein restent la première cause de mortalité liés aux cancers chez la femme et leur hétérogénéité induit un problème thérapeutique important. Dans ce contexte, les altérations de composition des ribosomes dans les cancers du sein ont été abordées dans des cohortes humaines et dans des modèles cellulaires de l’EMT (Transition Epithélio-Mésenchymateuse), un processus impliqué dans la tumorigenèse mammaire. Ces travaux ont permis d’identifier : i) deux facteurs impliqués dans la biogenèse des ribosomes, FBL (fibrillarine) et NCL (nucléoline) dont les variations d’expression sont associées à un mauvais pronostic chez les patientes ; et ii) des variations de composition du ribosome et de son activité traductionnelle dans l’EMT. L’ensemble de ces résultats soutient l’existence d’altérations de composition des ribosomes dans les cancers du sein

Ribosomes are responsible of translating mRNAs to proteins. Alterations of ribosome composition modify its translation activity and favour tumourigenesis. Identification of ribosomes composition alterations in breast cancers might correspond to a new mechanism responsible of mammary tumourigenesis and might open up novel therapeutic approaches. Indeed breast cancers represent the first cause of women mortality due to cancers and their heterogeneity induces an important therapeutic problem.In this context, alterations of ribosomes composition were determined in human cohorts and in EMT (Epithelial to Mesenchymal Transition) cellular models, the EMT being a process involved in mammary tumourigenesis. This studies identify : (i) two factors involved in ribosome biogenesis, FBL (fibrillarin) and NCL (nucleolin) whose expression variations are associated with poor prognosis in patients and (ii) variations of ribosome composition and its translational activity in EMT. Altogether, this data support the presence of ribosomes composition alterations in breast cancers, Les ribosomes sont responsables de la traduction des ARNm en protéines. Des modifications de la composition des ribosomes altèrent son activité de traduction et favorisent la tumorigenèse. L’identification des altérations de composition des ribosomes dans les cancers du sein pourrait être un nouveau mécanisme de tumorigenèse mammaire et ouvrir de nouvelles perspectives thérapeutiques. En effet, les cancers du sein restent la première cause de mortalité liés aux cancers chez la femme et leur hétérogénéité induit un problème thérapeutique important. Dans ce contexte, les altérations de composition des ribosomes dans les cancers du sein ont été abordées dans des cohortes humaines et dans des modèles cellulaires de l’EMT (Transition Epithélio-Mésenchymateuse), un processus impliqué dans la tumorigenèse mammaire. Ces travaux ont permis d’identifier : i) deux facteurs impliqués dans la biogenèse des ribosomes, FBL (fibrillarine) et NCL (nucléoline) dont les variations d’expression sont associées à un mauvais pronostic chez les patientes ; et ii) des variations de composition du ribosome et de son activité traductionnelle dans l’EMT. L’ensemble de ces résultats soutient l’existence d’altérations de composition des ribosomes dans les cancers du sein

The ribosome is responsible for the translation of mRNA into proteins. Within the ribosome, rRNAs play a crucial role in translation, and their post-transcriptional modifications regulate the ribosome’s translational activity and impact on gene expression. The human ribosome contains 106 2’-O-methylations added by fibrillarin (FBL). FBL functions through a box C/D snoRNP complex consisting of Nop56, Nop58 and 15.5kDa along with a box C/D small nucleolar RNA (snoRNA). The regulation of FBL ad the C/D box snoRNP complexe are unknown. This work exploitated strtuctural data on FBL and the methylation complex to build a model allowing the extrapolation of structure-function relationships. The impact of FBL acetylation was also investigated. 5-FU is a uracile analog, and its cytotoxicity depends mostly on its alteration of RNA metabolism. As such, 5-FU inhibits rRNA maturation and alters the localization of nucleolar factors such as FBL. 5-FU induced a novel FBL acetylation at position K292, decreased FBL interaction with the methylation complex proteins, and induced a large scale inhibition of its interactions. This discovered a new role of the deacetylase and the acetyltransferase CBP on snoRNP integrity. Moreover, this work suggests that these enzyme participate in the 5-FU-induced alteration of snoRNP. s. This work supports the involvement of post-translational modifications in the regulation of the rRNA C/D box snoRNP 2’-O-methylation complex, Le ribosome est responsable de la traduction des ARNm en protéines. Au sein du ribosome, les ARNr jouent un rôle central dans la traduction, et leurs modifications post-transcriptionnelles moduleraient l'activité traductionnelle du ribosome, impactant ainsi sur l'expression génique. Les ARNr humains contiennent 106 2'-O-méthylations, ajoutées par la fibrillarine (FBL). FBL fonctionne au sein d'un complexe snoRNP contenant les protéines Nop56, Nop58, 15.5kDa et un petit ARN nucléolaire (snoARN) à boîte C/D. La régulation de l'activité de la FBL et du complexe snoRNP à boîte C/D ne sont pas connus. Ce travail a exploité des données structurales de FBL et du complexe de méthylation pour construire un modèle permettant d'explorer les relations ses structure-fonction. L'impact de l'acétylation de FBL a également été exploré. Le 5-fluorouracile (5-FU) est un analogue de l'uracile, dont la cytotoxicité dépend de son altération du métabolisme des ARNr. Le 5-FU inhibe la maturation des ARNr et altère la localisation de plusieurs facteurs nucléolaires, dont FBL. Ce travail montre que le 5-FU induit une nouvelle acétylation de FBL en position K292. De plus, le 5-FU réduit l'association de FBL avec les membres protéiques du complexe de méthylation, et induit une baisse globale de ses interactions. De plus, ce travail propose un rôle nouveau de la déacétylase SIRT7 et de l'acétyltransférase CBP sur le complexe de méthylation. Ces enzymes semblent aussi participer aux dérégulations du complexe de méthylation induites par le 5-FU. L'ensemble de ces résultats supportent l'implication des modifications post-traductionnelles dans la régulation du complexe de méthylation des ARNr

5-Fluorouracil (5-FU) is an anti-metabolite intensely used in chemotherapeutic treatments in various cancers. The cellular and molecular mechanisms of action of this anti-cancer agent still remain to be determined. Because 5-FU is incorporated within all classes of RNA, knowledge of the different levels of gene expression regulation affected by 5-FU will help to decipher its mode of action. We hypothesized that the translational control is altered by 5-FU treatment as a consequence of disrupted RNA metabolism. In this study, the colorectal cancer cell line HCT116 has been treated or not by different doses of 5-FU for different periods of time to determine the time and dose window that induces modifications of cell behavior without leading to an extensive cell death. Translational reprogramming was then analyzed during this time and dose window. For this, cytoplasmic fractions were purified and separated through sucrose gradients to distinguish the actively translated mRNAs that are associated with polysomes from the inactive mRNAs associated with monosomes or free mRNAs. A microarray analysis was then performed to identify the mRNAs presented in monosome and polysome fractions with and without treatment. This polysome profiling approach reveals that 5-FU treatment did not turn-off the global translation efficiency, but rather modulates translation efficiency of specific mRNAs. Secondly, more than 640 mRNAs were found to be up-translated following 5-FU treatment. Finally, we have demonstrated that 5-FU induced up-regulation of HIVEP2 by a molecular mechanism involving an action of 5-FU on miR-155, Le 5-Fluorouracile (5-FU) est un anti-métabolite intensément utilisé dans les traitements chimio-thérapeutiques de nombreux cancers. Cependant, les mécanismes moléculaires de l'action de cet agent anti-cancer, son impact sur la biologie cellulaire et les processus de résistance restent encore largement à déterminer. Nous avons proposé que le 5-FU, en s'intégrant dans l'ARN induit une altération de la traduction. Dans cette étude, nous avons déterminé pour la lignée de cellules de cancer colorectal HCT116 la dose et le temps de traitement qui induit une modification du comportement cellulaire sans conduire à une mort cellulaire massive. Nous avons ensuite analysé le translatome de ces cellules traitées et celui de cellules non traitées. Pour cela, les fractions cytoplasmiques ont été purifiées et séparées sur gradients de saccharose pour séparer les ARNm qui sont associés aux polysomes de ceux associés aux monosomes et des ARN libres. L'analyse des modifications du translatome induites par le 5-FU montre que cette drogue est capable de stimuler la traduction d'un grand nombre d'ARNm qui codent pour des protéines possédant diverses fonctions. Cette activation traductionnelle est probablement médiée, au moins en partie, par une action du 5-FU sur les microARN. C'est ainsi que nous avons démontré que la stimulation de la traduction de l'ARNm du gène HIVEP2 est un mécanisme dépendant du microARN miR-155

Cancer cells produce large amounts of ribosomes to synthesize the proteins required for their rapid proliferation. The mechanisms leading to this increase in ribosome production are only partly understood, but they are related to the acquisition of the tumor phenotype. In addition, a new theory proposes that ribosomes are not neutral effectors of translation, but have a direct role in the regulation of gene expression. This theory is based on the observation that ribosome composition is heterogeneous in different cell types and according to environmental conditions. In this context, I have analyzed the relationships between changes in signals that control ribosome biogenesis, both quantitatively and qualitatively, and the development of the tumor phenotype. This manuscript reports three studies made during this PhD program. These studies identified: i) a novel regulator of the amount of ribosomes, the LN-Netrin-1 and ii) changes in the ribosome composition and function induced by genetic alterations (loss of activity of p53) and by the use of a chemotherapeutic molecule, the 5-Fluorouracil. These perturbations of the amount and the function of ribosomes modify the translation control and cell growth, cell proliferation and cell survival. From these results it can be conclude that ribosomes are elements involved in the regulation of gene expression and play a role in cancer pathology and response to chemotherapy, Les cellules cancéreuses produisent une grande quantité de ribosomes afin de synthétiser les protéines nécessaires à leur prolifération rapide. Les mécanismes qui conduisent à cette augmentation de la production de ribosome ne sont que partiellement compris, mais ils semblent intimement liés à l'acquisition du phénotype tumoral. De plus, une nouvelle théorie propose que les ribosomes ne sont pas des effecteurs neutres de la traduction, mais qu'ils jouent un rôle direct dans la régulation de l'expression génique. Cette théorie se base sur l'observation que la composition des ribosomes est hétérogène en fonction des types cellulaires et des conditions environnementales. Dans ce contexte, j'ai étudié les liens entre les altérations des signaux qui contrôlent la biogenèse des ribosomes, tant au niveau quantitatif que qualitatif, et le développement du phénotype tumoral. Ce manuscrit rapporte trois études effectuées au cours de mon travail de thèse. Ces études ont permis d'identifier : i) un nouveau régulateur de la quantité de ribosomes, la LN-Nétrine-1 et ii) des modifications de la composition et de la fonction des ribosomes induites par des altérations génétiques (perte d'activité de p53) et par l'utilisation d'une molécule chimiothérapeutique, le 5- Fluorouracile. Ces perturbations de la quantité et de la fonction des ribosomes modifient le contrôle de la traduction des cellules et la croissance, la prolifération et la survie cellulaire. Il ressort de ces résultats que les ribosomes sont des éléments qui participent au contrôle de l'expression génique et qui jouent un rôle dans la pathologie cancéreuse et la réponse au traitement chimiothérapeutique

Breast cancer is classified into several subtypes with varying clinical features. In particular, the so-called triple negative subtype is associated with a poor prognosis and a higher probability of recurrence. This is mainly due to the lack of specific targeted therapies. It is then necessary to identify new therapeutic targets in order to develop new drugs. Ribosome biogenesis is altered in most cancers due to the deregulation of the main oncogenic and tumor suppressor pathways, which regulate the activity of RNA Polymerase I, the enzyme responsible for ribosomal RNA transcription. Inhibition of ribosome biogenesis has been validated as a therapeutic tool, on the basis of three compounds that inhibit RNA Polymerase I activity (CX-5461, CX-3543 and BMH-21) that target cancer cells versus healthy cells. Their anti-cancer activity has been reported in several cancers, but the potential benefit of targeting ribosome biogenesis in certain types of breast cancer, including triple negative breast cancer, has not been evaluated yet. Although very little is known about ribosome biogenesis in triple negative breast cancer, there is now urgent need for new therapeutic tools and the potential of inhibiting ribosome biogenesis deserves to be evaluated. In addition, ribosome biogenesis depends on many maturation factors that represent a new family of targets to be explored. Here, the targeting of Fibrillarin is proposed. This may be relevant, as fibrillarin is often overexpressed in triple negative breast cancer and is associated with poor prognosis and risk of recurrence. Several cytotoxic mechanisms have been described in various cancer models upon treatment with RNA Pol I inhibitors and the elicited response appears to be model-specific. At present, since the effect on cells is unpredictable, the characterization of such a response in study models is not unfounded, especially since triple negative breast cancer is overly heterogeneous. The two main objectives of this thesis are: 1. To investigate the sensitivity of triple negative breast cancer lines to inhibition of ribosome biogenesis by pharmacological treatment targeting RNA Polymerase I or by genetic silencing of Fibrillarin; 2. To determine the mechanisms inducing the cytotoxic effect of inhibition of ribosome biogenesis in this model. In this work, using a large panel of cancer cell lines representing four different subtypes of triple negative breast cancer, it is shown that inhibition of ribosome biogenesis by BMH-21 and CX-5461 compounds induces strong cell growth inhibition and loss of the tumorigenic phenotype in vitro. In accordance with the heterogeneity of the model, triple negative cell lines have a wide range of sensitivity, but without association with subtypes according to common classification systems. Regarding induced cellular responses, an interesting phenomenon was observed: although BMH-21 causes apoptosis, no evidence of apoptosis was observed during treatment with CX-5461. In addition, it is demonstrated that inhibition of ribosome biogenesis downstream of RNA Polymerase I, by targeting Fibrillarin, also induces strong inhibition of cell growth, associated with an apoptotic response. Overall, these results suggest that inhibition of ribosome biogenesis could be a promising therapeutic strategy for triple negative breast cancer and validate Fibrillarin and ribosome RNA maturation as a new attractive therapeutic target; Le cancer du sein est classé en plusieurs sous-types dont les caractéristiques cliniques varient considérablement. En particulier, le sous-type dit triple-négatif est associé à un mauvais pronostic et à une probabilité plus élevée de récidive. Cela s'explique principalement par l'absence de thérapies ciblées spécifiques. Il est nécessaire d'identifier de nouvelles cibles thérapeutiques, afin de développer de nouveaux médicaments. La biogénèse des ribosomes est altérée dans la plupart des cancers, en raison de la dérégulation des principales voies oncogèniques et suppresseurs de tumeur, qui régulent l'activité de la ARN Polymerase I, l'enzyme responsable de la transcription de l'ARN ribosomique. L'inhibition de la biogénèse des ribosomes a été validée comme outil thérapeutique, à partir de trois composés qui inhibent l'activité de l'ARN Polymerase I (CX-5461, CX-3543 et BMH-21) qui ciblent les cellules cancéreuses par rapport aux cellules saines. Leur activité anti-tumorale a été rapportée dans plusieurs types de cancer, mais l'avantage potentiel de cibler la biogenèse des ribosomes dans certains types de cancer du sein, notamment les triple-négatifs, n'a pas été étudié à ce jour. Bien que très peu de choses soient connues sur la biogénèse des ribosomes dans le cancers du sein triple-négatifs, il y a aujourd'hui un besoin urgent de nouveaux outils thérapeutiques et le potentiel de l'inhibition de la biogenèse des ribosomes doit être évalué. Par ailleurs, la biogenèse des ribosomes dépend de nombreux facteurs de maturation qui représentent une nouvelle famille de cibles à explorer. Ici, le ciblage de la Fibrillarine est proposé. Cela pourrait être pertinent, car la fibrillarine est souvent surexprimée dans les cancers du sein triple-négatifs et est associée à un mauvais pronostic et à un risque de récidive. Plusieurs mécanismes cytotoxiques ont été décrits dans divers modèles de cancer lors du traitement par des inhibiteurs de l'ARN Pol I et la réponse obtenue semble être propre à chaque modèle. À l'heure actuelle, comme l'effet sur les cellules est imprévisible, la caractérisation d'une telle réponse dans les modèles d'études n'est pas sans fondement, d'autant plus que les cancer du sein triple-négatifs sont très hétérogènes. Les deux principaux objectifs de cette thèse sont donc : 1. Étudier la sensibilité de lignées de cancer du sein triple négatif à l'inhibition de la biogénèse des ribosomes par un traitement pharmacologique ciblant l'ARN Polymerase I ou bien par ciblage génétique de la Fibrillarine ; 2. Déterminer les mécanismes induisant l'effet cytotoxique de l'inhibition de la biogenèse des ribosomes dans ce modèle d'étude. Dans ce travail, en utilisant un large panel de lignées de cellules cancéreuses représentant quatre sous-types différents des triple-négatifs, il est montré que l'inhibition de la biogénèse des ribosomes par les composés BMH-21 et CX-5461 induit une forte inhibition de la croissance cellulaire et une perte du phénotype tumorigène in vitro. Conformément à l'hétérogéneité du modèle, les lignées cellulaires triple-négatives présentent une large gamme de sensibilité, mais sans association avec les sous-types selon les systèmes de classification courants. Concernant les réponses cellulaires induites, un phénomène intéressant a été observé : bien que le BMH-21 provoque l'apoptose, aucun signe d'apoptose n'a été observé lors du traitement par CX-5461. De plus, les résultats montrent que l'inhibition de la biogénèse des ribosomes en aval de l'ARN Polymerase I, par ciblage génique de la Fibrillarine, induit également une forte inhibition de la croissance cellulaire, associée à une réponse apoptotique. Dans l'ensemble, ces résultats suggèrent que l'inhibition de la biogénèse des ribosomes pourrait constituer une stratégie thérapeutique prometteuse pour le cancer du sein triple-négatif et positionnent la Fibrillarine et la maturation des ARN ribosomiques comme de nouvelles cibles thérapeutiques intéressante