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RESUME Les interventions ayant pour but de modifier le genome de la descendance sont proscrites depuis 1994 en France mais le developpement de methodes comme CRISPR-Cas9 conduit a s’interroger sur leurs utilisations potentielles sur les cellules germinales et l’embryon humains. La seule indication medicale acceptable serait d’eviter la transmission d’une pathologie genique a l’enfant mais les conditions, notamment celles relatives a l’efficacite et a l’innocuite de ces methodes, ne sont pas actuellement reunies pour envisager leur utilisation clinique. De plus il existe d’autres moyens permettant aux couples concernes de realiser leur projet parental. Les questions ethiques suscitees par ces technologies incitent a recommander l’ouverture d’une reflexion pluridisciplinaire qui devrait etre menee dans le cadre d’un debat plus large portant sur l’ensemble des interventions medicales realisees lors de de l’assistance medicale a la procreation, pouvant avoir des consequences sur le genome des enfants a naitre et eventuellement sur celui des generations suivantes. En revanche, les recherches, y compris sur les cellules germinales et l’embryon humains, devraient pouvoir etre menees quand elles sont scientifique- ment et medicalement justifiees.

RESUME Concernes par l’incidence croissante des cancers du sein, de la prostate et du testicule dans les pays industrialises, y compris en Europe, nous avons fait une revue de la litterature scientifique et des rapports francais sur les effets potentiellement carcinogenes des perturbateurs endocriniens (PEs) presents dans l’environnement et l’alimentation. Nous expliquons pourquoi il est tres difficile d’obtenir une preuve epidemiologique d’un effet cancerigene des PEs dans l’espece humaine, ce qui explique en partie les polemiques interminables sur cette question. Cependant les resultats d’une serie d’etudes independantes sont suffisants pour evoquer fortement un effet cancerigene de ces substances, notamment en cas de cancers hormono-dependants. Ces resultats proviennent d’etudes experimentales faites chez les rongeurs et des mesures de niveaux d’exposition dans le sang et les urines chez l’humain. La lecon de l’effet cancerigene transgenerationnel qui a ete observe apres traitement de femmes enceintes par le diethylstilbestrol et les resultats obtenus apres exposition experimentale de rongeurs au bisphenol A suggerent que les femmes enceintes et/ou allaitantes et les jeunes enfants doivent etre proteges en priorite. Alors qu’il a deja ete decide de reduire la presence de certains PEs comme les pesticides, les dioxines et les PCBs dans l’environnement, nous proposons de nouvelles mesures de precaution ciblees sur le bisphenol A et les phtalates qui sont notamment utilises dans le conditionnement des aliments. Cependant, avant d’interdire l’usage du bisphenol A dans les emballages alimentaires, il serait necessaire que les chercheurs industriels et academiques agissent de maniere concertee pour mettre au point des substituts au bisphenol A faisant preuve d’une meilleure innocuite.

RESUME La genetique moleculaire, discipline scientifique et medicale permettant l’analyse directe de l’ADN, est apparue dans les annees 80 et a connu un essor important grâce a la PCR (« Polymerase Chain Reaction ») qui a augmente considerablement la sensibilite des techniques. Les examens de genetique moleculaire, souvent qualifies de tests genetiques, ont de nombreuses applications medicales : diagnostic des maladies hereditaires a transmission mendelienne, diagnostics prenatal et preimplantatoire, determination des facteurs hereditaires de predisposition aux cancers, depistage neonatal, depistage des heterozygotes dans les familles de sujets atteints ou dans les populations a risque eleve, pharmacogenetique. En raison du nombre et de la complexite des facteurs susceptibles de moduler l’expression clinique de ces affections hereditaires, la realisation de ces examens doit etre accompagnee d’un conseil medical competent. Le determinisme d’un grand nombre de maladies communes dites multifactorielles releve de l’interaction de facteurs environnementaux, de facteurs comportementaux et de facteurs genetiques ; l’identification de ces derniers, a priori nombreux pour une meme maladie et entrainant chacun un risque relatif faible, est actuellement en cours par des etudes d’association (GWAS ou « Genome Wide Association Studies ») avec des resultats, jusqu’a present, limites ; differentes societes commerciales etrangeres proposent neanmoins de tels tests genetiques qui n’ont en fait aucun interet clinique reel. L’analyse du genome a des fins biometriques (empreintes genetiques) est en France strictement reglementee et comporte deux aspects : les applications judiciaires et les tests de paternite ; le recours aux tests de paternite pratiques a l’etranger dans des conditions techniques non controlees, le plus souvent via Internet, est devenu tres frequent. L’organisation et la reglementation de la genetique moleculaire en France sont detaillees ainsi que les questions posees par l’ensemble des tests genetiques pratiques : necessite de controles de qualite et d’un conseil medical pertinent, interdiction d’utiliser ces tests a des fins discriminatoires, en particulier en termes d’assurance et d’emploi, problemes poses par la diffusion des tests de convenance personnelle. Dans le cadre de la revision en cours des lois de bioethique, ce rapport formule plusieurs recommandations.

RESUME L’epissage alternatif est un processus qui permet, a partir d'une sequence genomique unique, de produire plusieurs ARN messagers correspondant a des proteines distinctes. Autrefois considere comme exceptionnel, l’epissage alternatif est maintenant la regle chez les organismes superieurs et notamment l’homme ou il constitue la base moleculaire de nombreuses pathologies. Il s’appuie sur un mecanisme tres flexible de selection des sites d’epissage qui implique la reconnaissance de sequences regulatrices sur l’ARN premessager par des proteines stimulatrices specifiques ou au contraire par des represseurs. Cette interaction ARN-proteine definit deux cibles therapeutiques potentielles qui ont ete inegalement explorees. La strategie de masquage des sequences regulatrices par des ARNs anti-sens etait la plus evidente et commence a donner lieu a des resultats interessants. Entierement nouvelle est celle que nous developpons actuellement et qui consiste a cibler les proteines activatrices par des molecules chimiques. A partir de la chimiotheque de l’Institut Curie, nous avons repere plusieurs de ces molecules, toutes appartenant a la famille des derives de l’indole, dont certaines sont capables d’inhiber les effets stimulateurs de plusieurs proteines activatrices individuelles.

RESUME L’objectif de cette revue est de souligner la multiplicite et l’importance, physiologique et pathologique, des niveaux de regulation de l’expression des genes qui interviennent posterieurement a l’etape d’initiation de la transcription. Celle-ci n’est en effet que le premier pas, certes decisif, d’une longue cascade d’evenements de regulation qui vont aboutir a la production selective des messagers fonctionnels appropries a la nature des cellules et a leurs besoins instantanes. Au travers de ce long parcours, dont seules quelques etapes seront etudiees ici, elle s’attachera a souligner le role central de l’ARN, non seulement comme substrat mais comme acteur de sa propre regulation. L’accent sera mis sur les liens tres nombreux existant avec la pathologie et jusqu’aux developpements therapeutiques dont l’ARN pourrait etre la cible privilegiee. Enfin, elle aura cherche a montrer la perennite des questions fondamentales posees depuis presque un demi-siecle et pour lesquelles la genomique aura fourni, a defaut de concepts nouveaux, la base de donnees et les outils indispensables pour entrer dans l’immense champ de la genomique fonctionnelle, seule capable d’apporter des reponses exploitables en therapeutique.

Some women with benign breast disease eventually develop breast cancer. The mammary gland undergoes tissue remodelling according to hormonal influences, involving a balance between quiescence, proliferation, and mechanisms of cell death. Proliferation and/or apoptotic events could therefore be investigated to help understand the mechanisms of benign lesion formation and identify mastopathies with a poor prognosis. bcl-2 expression was analysed by immunohistochemistry in 75 benign mastopathies. Protein levels were quantitated with an image analyser in various epithelial structures on frozen sections, including adenoses, fibroadenomas, ductal epithelial hyperplasias, cysts, and apparently normal surrounding lobules and ducts. bcl-2 levels were equivalent in apparently normal lobules and ducts, as well as in cysts and ductal hyperplasias. bcl-2 staining was significantly higher in fibroadenomas, known to be of lobular origin [mean = 10.1, quantitative immunochemistry score (QIC) arbitrary units (AU), n = 19], than in normal lobules (mean = 5.1 AU, n = 43, P = 7 x 10(-5). bcl-2 levels in normal lobules and ducts varied according to the menstrual cycle, being higher during the follicular than the luteal phase (P = 1.8 x 10(-2) and P = 1.7 x 10(-2), respectively). This was further supported by a statistical link (P = 5 x 10(-3) between high levels of circulating progesterone and weak bcl-2 staining in lobules and ducts. This progesterone-dependent variation was absent in fibroadenomas. No statistical correlation was found between bcl-2 expression and circulating levels of oestradiol, and follicle-stimulating or luteotrophic hormones. Although these are only preliminary results, they suggest an influence of progesterone on bcl-2 expression which might be lost in fibroadenomas. A hypothesis is proposed concerning the potential involvement of altered regulation of the apoptotic process in the formation of such benign lesions.

> L’epissage des genes est un passage quasi oblige de l’expression genique chez les eucaryotes superieurs. De plus, pour les trois quarts des genes de mammiferes [1], il existe des possibilites alternatives, parfois nombreuses, d’epissage de chaque transcrit primaire, ce qui rend compte au moins d’une grande partie de l’ecart entre le nombre de genes (environ 30 000) et celui des proteines qui est environ dix fois superieur [2]. L’epissage alternatif est donc un niveau essentiel de la regulation qualitative de l’expression des genes puisqu’il permet a une meme sequence d’ADN de produire des proteines differentes selon le tissu ou les conditions d’environnement. Mais, en contrepartie de son role createur de diversite proteique physiologique, l’epissage alternatif est aussi une occasion de produire des transcrits aberrants aux consequences pathologiques tres variees.

The U1 small nuclear ribonucleoprotein (snRNP) particle is one of the Sm class of snRNPs essential for splicing of precursor messenger RNA. Mammalian U1 snRNP contains a 165-nucleotide long RNA molecule and at least 11 proteins: the U1-specific 70K proteins A and C, and the common U snRNP proteins (B', B, D1, D2, D3, E, F and G). One of the functions of U1 snRNP is recognition of the 5' splice site, an event that requires both U1 RNA and U1 proteins. The 70K protein is the only heavily phosphorylated U1 protein in the cell. Isolated U1 snRNPs are associated with a kinase activity that selectively phosphorylates the 70K protein in vitro in a reaction requiring ATP. Here we investigate the role of phosphorylation of the 70K protein in the splicing of pre-mRNA. The 70K protein on U1 snRNPs was phosphorylated in vitro with either ATP, or with ATP-gamma S, which gave a thiophosphorylated product that was resistant to dephosphorylation by phosphatases. When HeLa nuclear splicing extracts that had been depleted of endogenous U1 snRNPs were complemented with U1 snRNPs possessing normal phosphorylated 70K protein, mature spliceosomes were generated and the splicing activity of the extracts was fully restored. By contrast, if thiophosphorylated U1 snRNPs were used instead, splicing was completely inhibited, although formation of the mature spliceosome was unaffected. Our data show that the state of phosphorylation of the U1-specific 70K protein is critical for its participation in a pre-catalytic step of the splicing reaction.

The extent and the variation of losses of genetic material were examined in a series of 191 human breast cancers by means of a set of 18 polymorphic DNA probes, specific of 7 chromosomal arms (1p, 1q, 3p, 11p, 13q, 17p and 18q) known to be frequently affected by allele losses. Frequencies of losses of heterozygosity ranged from a low of 3.5% (chromosome 13q) to a high of 27% (chromosome 3p). The number of sites involved in breast cancer added to the frequent occurrence of concomitant losses at several chromosomal arms within the same tumor suggest cooperative effects of these LOHs. We were therefore interested in assessing the existence of preferential associations between sets of LOHs in our panel of tumors. Statistically significant associations were found between LOHs at chromosomes Ip and 17p, and between LOHs at chromosomes 11p and 17p. Furthermore, since all the tumors presently studied had previously been analyzed for protooncogene amplification at 5 distinct chromosomal sites, we tested for associations between LOH and DNA amplification. Such associations were indeed observed as exemplified by the correlations observed between the LOH at 11p and amplification of the erbB2 gene and LOH at 17p and the amplification of the fig gene. The only correlation with clinicopathological parameters that could be observed linked the occurrence of LOHs on 11p with recurrent breast cancer (p = 0.015). Sets of several LOHs or LOHs and gene amplifications could not be significantly related to any marker of tumor aggressiveness. © 1992 Wiley-Liss, Inc.

Stable association of U2 snRNP with the branchpoint sequence of mammalian pre-mRNAs requires binding of a non-snRNP protein to the polypyrimidine tract. In order to determine how U2 snRNP contacts this protein, we have used an RNA containing the consensus 5' and the (Py)n-AG 3' splice sites but lacking the branchpoint sequence so as to prevent direct U2 snRNA base pairing to the branchpoint. Different approaches including electrophoretic separation of RNP complexes formed in nuclear extracts, RNase T1 protection immunoprecipitation assays with antibodies against snRNPs and UV cross-linking experiments coupled to immunoprecipitations allowed us to demonstrate that at least three splicing factors contact this RNA at 0 degree C without ATP. As expected, U1 snRNP interacts with the region comprising the 5' splice site. A protein of approximately 65,000 molecular weight recognizes the RNA specifically at the 5' boundary of the polypyrimidine tract. It could be either the U2 auxiliary factor (U2AF) (Zamore and Green (1989) PNAS 86, 9243-9247), the polypyrimidine tract binding protein (pPTB) (Garcia-Blanco et al. (1989) Genes and Dev. 3, 1874-1886) or a mixture of both. U2 snRNP also contacts the RNA in a way depending on p65 binding, thereby further arguing that the latter may correspond to the previously characterized U2AF and pPTB. Cleavage of U2 snRNA sequence by a complementary oligonucleotide and RNase H led us to conclude that the 5' terminus of U2 snRNA is required to ensure the contact between U2 snRNP and p65 bound to the RNA. More importantly, this conclusion can be extended to authentic pre-mRNAs. When we have used a human beta-globin pre-mRNA instead of the above artificial substrate, RNA bound p65 became precipitable by anti-(U2) RNP and anti-Sm antibodies except when the 5' end of U2 snRNA was selectively cleaved.

The advent and recent use of Genome-Wide Association studies (GWAS) for the search of genetic predisposition markers for prostate cancer since 2006 has put a very strong emphasis on the 8q24 locus where several single nucleotide polymorphisms (SNP) have been significantly associated with an increased relative risk. A wealth of recent papers have all confirmed the interest of this locus and identified several others. Interestingly, these markers seem to have additive effects pointing to the high complexity of prostate cancer predisposition. This situation along with our current inability to identify any causal gene(s) in these regions make these findings difficult to translate into routine clinical practice at the present time, a fortiori in terms of population screening.

The aim of this review is to draw the attention to the numerous steps of gene expression which operate at the RNA level and which are significant drivers of the transformation process. The analysis of genomic abnormalities was at first limited to gross chromosomal alterations and to DNA point mutations. Then came the era of transciptomes which were originally believed, since they were mRNAs, to be a faithful reflection of the expressed proteins. As a matter of fact, this first-generation transcriptomics gave only a global, quantitative, assessment of gene expression at a given locus but overlooked the qualitative diversity of the messenger population generated by alternative splicing. We will show that beyond their essential role in the regulation of functions specific to metazoan like development, alternative splicing brings about an important vulnerability to mutations which is at the origin of many pathologies including cancer. The second aspect covered by this review is that of a rather novel category of RNAs, the microRNAs which, although non-coding, functionally behave as oncogenes or tumor suppressors through the negative control they exert on conventional oncogenes or suppressors. Beyond their diagnostic and prognostic interest, these two mechanisms offer entirely novel potential therapeutic targets.

Nuclear Organisation and Subnuclear Bodies.- Searching for Active Ribosomal Genes.- Toxic RNA in the Nucleus: Unstable Microsatellite Expression in Neuromuscular Disease.- Assembly and Traffic of Small Nuclear RNPs.- Intranuclear Pre-mRNA Trafficking in an Insect Model System.- Photobleaching Microscopy Reveals the Dynamics of mRNA-Binding Proteins Inside Live Cell Nuclei.- Imaging of Single mRNAs in the Cytoplasm of Living Cells.

Resume Cette revue retrace la decouverte des oncogenes qui sont les avatars actives de proto-oncogenes cellulaires normaux et indispensables aux fonctions de proliferation et de differenciation et a leur controle. Leur activation perturbe le mecanisme normal de ce controle assure par une balance subtile de leurs effets et de ceux des anti-oncogenes ou genes suppresseurs de tumeurs. On peut raisonnablement esperer que la connaissance du profil complet des alterations de ces deux types de genes essentiels dans la tumeur ou chez l'individu cliniquement sain permette un apport decisif a l'evaluation respectivement du pronostic et de la predisposition hereditaire.

In breast cancer, axillary lymph node invasiveness is the major prognostic factor in predicting relapse and metastasis. Nevertheless, since 30% of node-negative tumors also relapse, it is necessary to develop other independent prognostic factors. Oncogene amplification and the level of cathepsin D (cath-D), an acidic lysosomal protease produced and secreted in excess by breast cancer cells, have been proposed as additional prognostic factors. We have compared the cytosolic cath-D level and the amplification of three oncogenes: c-myc, neu-erb-B-2 and int-2 in 140 primary breast carcinomas and 64 axillary lymph nodes collected in 1987 and 1988 at the Cancer Center of Montpellier (Centre Paul Lamarque). None of the patients had previously received hormonal or chemotherapy. The cath-D concentration was measured with an immunoradiometric assay using monoclonal antibodies. DNA purified from the same samples was analyzed by a standard Southern blotting technique to estimate oncogene amplification. No correlation was found between the level of cath-D in the tumor and node invasiveness. Using a cut-off level of 60 pmol/mg protein, the status of cath-D was not correlated with neu-erb-B-2 and int-2 amplification and only correlated with c-myc amplification (P = 0.011). Both c-myc and cath-D are associated with cell proliferation, induced by estrogens in ER+ breast cancer, and constitutively produced in ER- breast cancer. The level of cath-D was significantly higher in the invaded lymph nodes (P = 0.04) than in the histologically non-invaded ones. Nevertheless, some non-invaded lymph nodes contained a high level of cath-D, as confirmed by immunoperoxidase staining. In conclusion, in breast cancer, a high cytosolic cath-D concentration is more frequent in tumors with c-myc amplification but is dissociated from neu-erb-B-2 or int-2 amplification, suggesting that the determination of these three markers will have an additional prognostic value.

Methods and Platforms for the Quantification of Splice Variants' Expression.- Pre-mRNA Missplicing as a Cause of Human Disease.- Alternative Splicing: Therapeutic Target and Tool.- SR Proteins as Potential Targets for Therapy.- Misregulation of Tau Alternative Splicing in Neurodegeneration and Dementia.- Spinal Muscular Atrophy and Therapeutic Prospects.- Misregulation of Alternative Splicing Causes Pathogenesis in Myotonic Dystrophy.- Redirecting Splicing to Address Dystrophin Mutations: Molecular By-pass Surgery.- Altered Splicing in Prelamin A-Associated Premature Aging Phenotypes.- Splicing Modulation as a Modifier of the CFTR Function.

The prevalence of alternative splicing as a target for alterations leading to human genetic disorders makes it highly relevant for therapy. Here we have usedin vitrosplicing reactions with different splicing reporter constructs to screen 4,000 chemical compounds for their ability to selectively inhibit spliceosome assembly and splicing. We discovered indole derivatives as potent inhibitors of the splicing reaction. Importantly, compounds of this family specifically inhibit exonic splicing enhancer (ESE)-dependent splicing, because they interact directly and selectively with members of the serine-arginine-rich protein family. Treatment of cells expressing reporter constructs with ESE sequences demonstrated that selected indole derivatives mediate inhibition of ESE usagein vivoand prevent early splicing events required for HIV replication. This discovery opens the exciting possibility of a causal pharmacological treatment of aberrant splicing in human genetic disorders and development of new antiviral therapeutic approaches.

The spliceosome is a dynamic and flexible ribonucleoprotein enzyme that removes intronic sequences in a regulated manner. Spliceosome action enables one stretch of genomic DNA sequence to yield several mRNAs that encode different proteins. It depends on a flexible mechanism for selecting splice sites, which calls for regulatory sequences (splicing enhancers or silencers) recognized by cognate trans-acting protein factors and constitutive ribonucleoprotein devices to build up the catalytic core. The identification of both types of elements now offers a comprehensive insight into how the spliceosome is adapted to carry out the removal of different introns and suggests novel therapeutic targets to, ultimately, restore a physiological pattern of alternatively spliced variants in a large repertoire of pathologies.

Chromatin Remodeling Factors and DNA Replication.- Epigenetic Inheritance of Chromatin States Mediated by Polycomb and Trithorax Group Proteins in Drosophila.- How to Pack the Genome for a Safe Trip.- Chromatin Modifications on the Inactive X Chromosome.- Chromatin Mechanisms in Drosophila Dosage Compensation.- DNA Methylation in Epigenetic Control of Gene Expression.- The Epigenetic Breakdown of Cancer Cells: From DNA Methylation to Histone Modifications.- Developmental Regulation of the ?-Globin Gene Locus.- Epigenetic Regulation of Mammalian Imprinted Genes: From Primary to Functional Imprints.- Seed Development and Genomic Imprinting in Plants.

Spontaneous apoptosis by in situ detection of DNA fragmentation (DNAf) was investigated in breast invasive ductal carcinoma (IDC) frozen samples removed from 61 untreated patients. The incidence of DNAf was low in carcinoma cells and was mainly detected in the stroma. In the stroma at a distance from carcinoma cells, DNAf was inversely related to estradiol plasma level variations (p=0.01), indicating that it probably remained under physiological hormonal regulation. In the stroma adjacent to carcinoma cells, DNAf was correlated to tumor progression parameters such as the presence of a comedo intra ductal carcinoma (DCIS) component (p=0.001) and axillary lymph node metastasis (p=0.002), suggesting that this stromal compartment more probably represented a tumoral component closely associated to epithelial tumor cells. Therefore, the detection of DNAf in the adjacent stroma of breast carcinoma could help to predict progression in non invasive tumors and also in invasive tumors in those patients without lymph node invasion.

Prediction and Statistical Analysis of Alternatively Spliced Exons.- Multiple Roles of the SR Protein Family in Splicing Regulation.- Heterogeneous Nuclear Ribonucleoprotein Particle A/B Proteins and the Control of Alternative Splicing of the Mammalian Heterogeneous Nuclear Ribonucleoprotein Particle A1 Pre-mRNA.- Phosphorylation-Dependent Control of the Pre-mRNA Splicing Machinery.- Splicing Regulation in Drosophila Sex Determination.- Alternative Pre-mRNA Splicing and Regulation of Programmed Cell Death.- Alternative Pre-mRNA Splicing and Neuronal Function.- Modulation of Alternative Splicing by Antisense Oligonucleotides.

The purpose of this review is to emphasize the multiplicity and importance, physiological and pathological, of gene regulation levels which operate after the initiation step of transcription. Albeit crucial, this step is only the first one of a long cascade of events which eventually end up in the selection of functional messengers appropriate to the nature of the cells and to their immediate needs. Throughout this long pathway, of which only a few steps will be mentioned here, this review will attempt to address the central role played by RNA, not only as a substrate but as an actor of its own regulation. Emphasis will be put on the numerous connections with pathology up to the development of new therapeutics specifically targeting RNA. It highlights the perennity of basic questions, nearly half a century old, for which genomics, short of new concepts, will provide the databases and tools required to access to the immense field of functional genomics, the only one likely to bring relevant answers for therapeutics.

Rho Family Proteins and Regulation of the Actin Cytoskeleton.- 1 Introduction.- 2 Structure and Regulation of Rho Family Proteins.- 2.1 Primary Structure of Rho Family Proteins.- 2.2 Expression of Rho Family Proteins.- 2.3 Post-Translational Modifications.- 2.4 Structural Analysis of Rho Family Proteins.- 3 Rho Family Proteins and Growth Factor-Induced Actin Reorganization.- 3.1 Rho.- 3.2 Rac.- 3.3 Cdc42.- 3.4 Other Rho Family Proteins.- 3.5 Links Between Different Rho Family Proteins:A Role in Cell Migration?.- 4 Rho Family Proteins and Cell Adhesion.- 4.1 Adhesion to the Extracellular Matrix.- 4.2 Intercellular Adhesions.- 5 Rho Family Proteins and Cytokinesis.- 6 Other Responses Regulated by Rho Family Proteins.- 6.1 Activation of the NADPH Oxidase and Phagocytosis.- 6.2 Secretion and Endocytosis.- 6.3 Mitogenesis and Transformation.- 7 Conclusions.- References.- Regulation of Cytoskeleton and Cell Adhesion by Rho Targets.- 1 Introduction.- 2 Regulation of Rho Activity.- 3 Functions of Rho.- 3.1 Formation of Stress Fiber and Focal Adhesion.- 3.2 Smooth Muscle Contraction.- 3.3 Neurite Retraction.- 3.4 Cytokinesis.- 3.5 Cell-Cell Adhesion.- 3.6 Actin Filaments Beneath Plasma Membrane.- 3.7 Other Functions.- 4 Rho Targets.- 5 Functions of Rho Targets.- 5.1 Rho-Kinase and MBS.- 5.2 mDia and Bnil.- 5.3 Other Rho Targets.- 6 Conclusion.- References.- Rnd Proteins: A New Family of Rho-Related Proteins That Interfere with the Assembly of Filamentous Actin Structures and Cell Adhesion.- 1 Introduction.- 2 Characterisation of Three New Members of the Rho Family.- 3 Biochemical Properties of Rndl: G-Proteins That Do Not Switch?.- 4 Expression in Tissues and Localization of Rndl in the Brain.- 5 Effects of Rndl Expression on the Formation of Actin Stress Fibers.- 6 Localization of Rndl in Swiss 3T3 Fibroblasts and Microinjected MDCK Cells.- 7 A Role for Rnd Proteins in Transformation?.- 8 Rnd Proteins and Targets of Farnesyl-Transferase Inhibitors.- 9 Conclusions.- References.- The DH Protein Family, Exchange Factors for Rho-Like GTPases.- 1 Introduction.- 1.1 Small GTPases.- 1.2 Rho-Like GTPases.- 2 Identification of DH Proteins, GEFs for Rho-Like GTPases.- 3 Structure and Function of DH Proteins.- 3.1 Catalytic Function.- 3.2 Functional Domains in DH Proteins.- 3.2.1 DH and PH Domains.- 3.2.2 Other Domains in DH Proteins.- 4 Cellular Functions of DH Proteins.- 4.1 Oncogenic Transformation.- 4.2 Invasion.- 4.3 Neuronal Development.- 4.4 Development.- 4.5 Modulation of RasGEF Activity.- 5 DH Proteins in Signaling Pathways.- 5.1 Activation of GEF Activity by Phosphorylation.- 5.2 Activation of Rho-like GTPases by Membrane Localization of GEFs.- 5.3 Signal Mediation by DH Proteins in Protein Complexes.- 5.4 Role of DH Proteins in Ras Transformation.- 6 Concluding Remarks.- References.- RhoGAPs and RhoGDIs (His)stories of Two Families.- 1 RhoGDI (GDP Dissociation Inhibitors): Who Needs Them?.- 1.1 A Family of RhoGDIs.- 1.2 Biochemical Properties.- 1.3 From Structure to Function: Insights Deduced from RhoGDI-1 3-D Structure.- 1.4 In Vivo Functional RhoGDI as a Specific Tool for Gaining Insight into Rho Function.- 1.5 Cellular Localisation.- 1.6 A Hematopoietic RhoGDI, RhoGDI-2 (Ly/D4-RhoGDI) Could Play a Role During Apoptosis.- 1.7 A Third RhoGDI Exhibits Catalytic Specificity and Cytoskeleton Localisation.- 2 GTPase Activating Proteins (GAPs) for Rho-Like GTPases: A Catalytic Module Found Within a Plethora of Multifunctional Proteins.- 2.1 GAP Activity and Identification of p50-RhoGAP.- 2.2 Birth of the Rho-GAP Family of Proteins.- 2.3 Bcr (Breakpoint Cluster Region) Gene Product.- 2.4 Abr (Active Bcr-Related) Gene Product.- 2.5 The Chimaerin Family of Proteins (?l, ?2, ?1, and ?2).- 2.6 p85? and p85? Subunits of PI3-Kinase.- 2.7 p190 (p120-RasGAP-Associated Protein) and p190b.- 2.8 Other Mammalian Rho-GAPs: 3BP-1, RLIP76, Myr5, Myosin-IXb, p122-Arp, p115 and p58-Mgc.- 2.9 Non-Mammalian Rho-GAPs: Graf, RotundRacGAP, CeGAP, DdRacGAP, Bem2, Bem3, Dbml, LRG1 and RGA1.- 3 Conclusions.- 3.1 Too Many Rho GTPase-Regulating Factors?.- 3.2 A Final Word.- References.- Roles of PAK Family Kinases.- 1 Introduction.- 2 Distribution of PAK Family Kinases.- 3 Regulation of PAK Activity.- 4 Functions of Fungal Ste20p/PAK-Like Kinases.- 5 PAKs and Phosphorylation Pathways.- 6 PAK and Cellular Transformation.- 7 Morphological Roles for PAK Downstream of Cdc42 and Rac.- 8 Activities Associated with the Various Domains of PAK.- 8.1 Proline-Rich Domains.- 8.2 The p21-Binding Domain.- 8.3 The Auto-Regulatory Domain.- 8.4 The Kinase Domain.- 9 Conclusions.- References.- Rac and Cdc42 Effectors.- 1 Introduction.- 2 Effector Proteins of Rac and Cdc42.- 2.1 Potential Effectors of Rac and Cdc42 Involved in Cytoskeletal Rearrangements.- 2.2 Potential Effectors of Rac and Cdc42 Induced Transcriptional Activation.- 2.3 Potential Effector Pathways of Rac and Cdc42 Involved in Cell Growth Control.- 2.4 Phox 67, a Rac Target in the Superoxide Formation of Phagocytic Cells.- 3 Concluding Remarks.- References.- Small GTPases of the Rho Family and Cell Transformation.- 1 Introduction.- 1.1 The Rho Family.- 1.2 Regulators of the Rho Family and their Oncogenic Properties.- 2 Rho Proteins and Cell Transformation.- 2.1 Intrinsic Transforming Properties of Rho Proteins.- 2.2 Cross-Talk Between Rho Controlled Pathways.- 3 Cross-Talk Between Ras and Rho-Dependent Pathways in Cell Transformation.- 3.1 Cooperation Between Ras and Rho Pathways.- 3.2 Cooperation Between Raf and Rho Pathways.- 4 Signalling Pathways Downstream of Rho Proteins Involved in Cell Transformation.- 4.1 Rho-Dependent Activation of the JNK/SAPK Pathway.- 4.2 The Cytoskeleton and Rho Proteins.- 5 Rho Proteins and Apoptosis.- 6 Concluding Remarks.- References.- Rho GTP-Binding Proteins as Targets for Microbial Pathogens.- 1 Introduction.- 2 Microbial Pathogens and the Epithelial Cell Actin Cytoskeleton.- 2.1 Microbial Pathogens and Actin.- 2.2 Rho GTP-Binding Proteins.- 3 Interaction of Bacterial Protein Toxins with the Rho Subfamily of GTP-Binding Proteins.- 3.1 Toxins Inhibiting Rho.- 3.2 Toxins Activating Rho.- 4 Invasive Bacterial Factors and the Rho Subfamily of GTP-Binding Proteins.- 4.1 Bacterial Effectors of Salmonella or Shigella-Induced Internalization by Epithelial Cells.- 4.2 Cdc42 Is Involved in Salmonella Entry, Whereas Rho Proteins Are Required for Shigella Entry.- References.- Rho GTPases in Development.- 1 Introduction.- 2 The Rho Family of Small GTPases.- 2.1 Rho-Mediated Signal Transduction.- 2.2 Genetic Analysis of Signal Transduction in Simple Organisms.- 3 Genetic Analysis of Rho GTPases in Drosophila Development.- 3.1 Expression of Rho Family GTPases in Drosophila Embryogenesis.- 3.2 Oogenesis.- 3.3 Gastrulation.- 3.4 Dorsal Closure.- 3.5 Tissue Polarity.- 3.6 Eye Development.- 3.7 Neural Development.- 3.8 Muscle Development.- 4 Rho Family GTPases in C. elegans.- 4.1 Conservation of Rho GTPases and their Regulators and Targets in Worms.- 4.2 Embryo Elongation.- 4.3 Neural Development.- 5 Rho GTPase Function in Mammals.- 5.1 Knockout Mice.- 5.2 Transgenic Mice.- 5.3 Diseases of Human Development.- 6 Summary.- References.- Erratum.

TOP Genes: A Translationally Controlled Class of Genes Including Those Coding for Ribosomal Proteins.- 1 Introduction.- 2 TOP Genes and TOP mRNAs.- 2.1 How Many TOP Genes?.- 2.2 How Much TOP mRNA?.- 3 Phenomenology of the Translational Regulation of TOP mRNAs.- 3.1 General and Specific Regulation of Protein Synthesis.- 3.2 Translational Regulation of TOP mRNAs.- 4 Some Features of TOP mRNA and of Its Translational Regulation.- 4.1 TOP mRNAs Are Capped.- 4.2 TOP mRNAs Extracted from Inactive mRNPs Are Translatable.- 4.3 Bimodal Distribution of TOP mRNA.- 4.4 Quantitative Differences in Polysome Association of Different TOP mRNAs.- 4.5 Relocation of TOP mRNA Between Polysomes and mRNPs Is Reversible and Fast.- 5 Mechanism of Translational Regulation of TOP mRNAs.- 5.1 Translational Regulation of TOP mRNA Does Not Involve a Feedback Inhibition by the Translation Product.- 5.2 Effect of the Amount of Ribosomes on TOP mRNA Translation.- 5.3 The 5'UTR is the cis -Acting Element of the Translational Control.- 5.4 Some Proteins Interact with the 5'UTR of TOP mRNAs.- 5.5 Translational Regulation of TOP mRNA is Not Due to Limiting Amounts of eIF-4E.- 5.6 Effect of the Phosphorylation State of Ribosomal Protein S6 on Translational Regulation of TOP mRNA.- 5.7 Global vs. TOP mRNA Translational Regulation.- References.- RNase L: Effector Nuclease of an Activatable RNA Degradation System in Mammals.- 1 Introduction.- 2 The Interferons.- 3 The 2-5A Pathway.- 3.1 The 2-5A Synthetases.- 3.2 2-5A.- 4 RNase L.- 4.1 Detection of RNase L.- 4.2 Subcellular Localization of RNase L.- 4.3 Structure of RNase L.- 4.4 RNase L Activity.- 5 RLI.- 6 Conclusion.- References.- 3' Untranslated Regions of c-myc and c-fos mRNAs: Multifunctional Elements Regulating mRNA Translation, Degradation and Subcellular Localization.- 1 c-fos and c-myc mRNA Degradation and Translation.- 1.1 3'UTR and mRNA Decay.- 1.2 3'UTR and mRNA Translation.- 1.3 Is There a Link Between Translation and 3'UTR-Directed Degradation?.- 2 Localization of mRNAs in the Cytoplasm and Their Association with the Cytoskeleton.- 2.1 Localization of mRNAs.- 2.2 Association of mRNAs and Polysomes with the Cytoskeleton: Cytoskeletal-Bound Polysomes.- 2.3 Cytoskeletal-Bound Polysomes Are Enriched in Specific mRNAs Including c-myc and c-fos.- 2.4 Targeting of c-myc to the Cytoskeletal and the Perinuclear Cytoplasm: Role of the 3'UTR.- 2.5 Localization Signals in c-myc mRNA.- 3 Summary and Future Perspectives.- References.- Cell-Free Systems for Analysis of Cytoplasmic mRNA Turnover.- 1 Introduction.- 2 mRNA Decay in Lower Eukaryotes.- 2.1 Tetrahymena thermophila.- 2.2 Yeast.- 3 Viral Induction of mRNA Decay.- 3.1 Herpes simplex Virus Type 1 (HSV-1).- 3.2 Human Papillomavirus Type 16 (HPV-16).- 4 mRNA Stability in Plants.- 4.1 Spinach Chloroplasts.- 4.2 Soybean.- 5 Xenopus.- 5.1 Albumin mRNA Decay in Xenopus Liver.- 5.2 Xenopus Oocyte mRNAs.- 5.2.1 Maternal mRNA Deadenylation.- 5.2.2 Maternal Xlhboxl mRNA Degradation.- 6 Chicken.- 7 Mammalian mRNA Turnover.- 7.1 Development of Functional In Vitro Decay Systems.- 7.2 Characterization of Decay Pathways.- 7.2.1 H4 Histone mRNA Decay.- 7.2.2 c-myc mRNA Decay.- 7.3 Identification/Purification of mRNases.- 7.3.1 Histone mRNA 3'=>5' Exoribonuclease.- 7.3.2 Poly(A) Exoribonuclease from HeLa Cells.- 7.3.3 5'=>3' Exoribonuclease in Mouse Sarcoma Ascites Cells.- 7.3.4 RNase L.- 7.4 Identification/Purification of trans-Acting Decay Regulators.- 7.4.1 c-myc mRNA Decay.- 7.4.2 Poly(A)-Binding Protein (PAB) and mRNA Stability.- 7.4.3 GM-CSF mRNA Turnover.- 7.4.4 ss -Globin mRNA Stability.- 7.5 Identification/Function of cis -Acting Instability Determinants.- 7.5.1 Insulin-Like Growth Factor I (IGF-I) mRNAs.- 7.5.2 A+ U-Rich Sequences.- 7.5.3 c-myc 3'UTR and Coding Region.- 7.5.4 Ribonucleotide Reductase R2 mRNA 3'UTR.- 7.6 Regulation of mRNA Decay.- 7.6.1 Histone Autoregulation.- 7.6.2 Stability of Urokinase-Type Plasminogen Activator (uPA) mRNA.- 7.6.3 Decay of Manganese Superoxide Dismutase (MnSOD) mRNAs.- 7.6.4 Stabilization of Transforming Growth Factor ssl (TGF-ssl) mRNAs.- 8 Conclusion.- References.- Mechanisms for Posttranscriptional Regulation by Iron-Responsive Elements and Iron Regulatory Proteins.- 1 Introduction.- 2 The Iron-Responsive Element (IRE): A Cis -Regulatory RNA Element.- 3 Iron-Regulatory Proteins (IRPs).- 4 Translational Control of IRE Containing RNAs by IRE/IRP Interactions in the 5'UTR.- 5 Control of Transferrin Receptor mRNA Stability by IRE/IRP Interactions in the 3'UTR.- 6 "Iron-Responsive" Elements Also Respond to Other Cellular Signals.- 7 Summary and Perspectives.- References.- Interaction Between Iron-Regulatory Proteins and Their RNA Target Sequences, Iron-Responsive Elements.- 1 Introduction.- 2 Biochemistry of IRP-1.- 2.1 Modulation of IRP-1 Binding Activity.- 2.2 RNA-Binding Domain of IRP-1.- 3 Evidence for a Second IRE-Binding Protein.- 4 Characterisation of IRP-2.- 4.1 Differential Iron Regulation of IRP-1 and IRP-2.- 5 Sequence and Structure of Iron-Responsive Elements.- 6 Role of IRE Loop Structure in Protein Recognition...- 7 IRP-1 and IRP-2 Bind Distinct Sets of RNA Targets.- 8 Summary.- References.- Cytoplasmic Fate of Eukaryotic mRNA: Identification and Characterization of AU-Binding Proteins.- 1 Introduction.- 2 Stable mRNAs.- 3 Unstable mRNAs.- 4 Cis -Acting Elements.- 4.1 ARE Sequence Requirements.- 4.2 Genomic Rearrangements of the ARE.- 4.3 Other Sequences.- 5 Translational Dependence of ARE-mediated mRNA Turnover.- 6 Poly(A) Shortening in ARE mRNA Decay.- 7 Subcellular Localization of mRNA Decay.- 8 How Might the ARE Be Recognized?.- 8.1 Primary Structure.- 8.2 Secondary Structure.- 9 Trans -Acting Factors.- 9.1 AUBF.- 9.2 p32 and hnRNP AO.- 9.3 hnRNPs.- 9.4 AUF-1.- 9.5 AU-A, AU-B, and AU-C.- 9.6 AU-H.- 9.7 Hel-Nl.- 10 Functions.- 11 Posttranslational Modifications.- 11.1 Phosphorylation.- 11.2 Metal Ions.- 11.3 Methylation.- 11.4 Redox State.- 12 Purification.- 13 Cloning.- 14 Sequence Information.- 15 ARE-Mediated Decay Model.- References.- Translational Control by Polyadenylation During Early Development.- 1 Introduction.- 2 Biological Importance of Translational Control in Development.- 3 Translational Repression in Oocytes.- 4 Control of Polyadenylation During Oocyte Maturation.- 4.1 Cytoplasmic Polyadenylation: Identification of Cis Elements.- 4.2 Identification of Proteins Involved in Cytoplasmic Polyadenylation.- 4.3 Deadenylation in Maturing Oocytes.- 5 Control of Polyadenylation in Eggs and Embryos.- 5.1 Polyadenylation in Embryos.- 5.2 Embryo-Specific Deadenylation.- 6 Conclusion.- References.- Function and Characterization of Poly(A)-Specific 3' Exoribonucleases.- 1 Introduction.- 2 Biochemistry of mRNA Polyadenylation.- 2.1 Polyadenylation in Mammalian Cells.- 2.1.1 Nuclear Polyadenylation.- 2.1.2 Cytoplasmic Polyadenylation.- 2.2 Polyadenylation in Yeast.- 3 The Functional Importance of RNA Poly(A) Tails ...- 3.1 Poly (A) Tail Shortening and RNA Decay.- 3.2 The Involvement of mRNA Poly(A) Tails During Translation.- 4 Poly(A)-Specific 3' Exoribonucleases.- 4.1 Mammalian Nucleases.- 4.2 Plant Nucleases.- 4.3 Trypanosomatid Nucleases.- 4.4 Yeast Nucleases.- 4.5 Bacterial Nucleases.- 5 Regulation of Poly (A) Tail Shortening.- 6 Summary and Perspectives.- References.

To study the role of ras, p53 genes and HPV virus (16 and 18) in the development of prostate cancer, we analyzed tissue sections from 27 patients affected with carcinomas (stages A to D) and from 24 patients with adenomas. Mutations of H, K and N-ras and p53 (exons 2-9) were studied by SSCP and DNA sequencing. Accumulation of p53 protein was studied by immunohistochemistry on tissue sections. Tumors were also analyzed for the presence of HPV16 and -18 sequences by PCR and DNA hybridization with sequence-specific oligonucleotides. No mutation was found in the three ras genes studied, either in carcinomas or adenomas. By SSCP analysis we identified p53 mutations in only 2 of 19 carcinomas studied, both in exon 7. Immunohistochemical results strongly correlate with the SSCP results: p53 protein was positive in tumors with p53 mutation but not in others; 32% of studied adenomas had detectable HPV16 DNA, while 53% of carcinomas were HPV16+. Among these I presented a p53 mutation. No HPV18 E6 sequence could be detected. Our data show that in prostate tumors from France, mutations of p53 and ras are rare events but that these tumors display detectable HPV16 DNA at a high frequency. The low incidence of p53 mutation, associated to a significant proportion of tumors showing HPV16 DNA, could suggest that in prostate cancer HPV16 infection could participate in p53 inactivation by E6.

Proteins constitute the working-class molecules of the cell. Hence, understanding the way they act is a prerequisite for understanding how a cell functions and how life evolves. Aspects such as the protein-ligand relationship, recognition, protein evolution by point mutation, enzyme-substrate interactions, behaviour of an enzyme in a living cell, control and dynamics of enzyme networks as well as the physico-chemical background of enzyme actions and multi-enzyme complexes are comprehensively treated in this volume.

International audience; Cellular transition from the resting state to DNA synthesis involves master switches genes encoding transcriptional factors (e.g., fos, jun, and egr genes), whose targets remain to be fully characterized. To isolate coding sequences specifically accumulated in late G1, a differential screening was performed on a cDNA library prepared from hamster lung fibroblasts stimulated for 5 h with serum. One of the positive clones which displayed a sevenfold induction, turned out to code for a protein sharing homology to Ras-like products. Cloning and sequence analysis of the human homolog revealed that this putative new small GTPase, referred to as rhoG, is more closely related to the rac, CDC42, and TC10 members of the rho (ras homolog) gene family and might have diverged very early during evolution. rhoG mRNA accumulates in proportion to the mitogenic strength of various purified growth factors used for the stimulation, as a consequence of transcriptional activation. G1-specific RNA accumulation is impaired upon addition of antimitogenic cyclic AMP and is enhanced when protein synthesis is inhibited, mainly as a result of RNA stabilization. rhoG mRNA expression is observed in a wide variety of human organs but reaches a particularly high level in lung and placental tissues.

We report here that transport of the protein product of the c-fos proto-oncogene from the cytoplasm, where it is synthesized, into the nucleus, where it operates as part of the AP-1 transcription complex, is not spontaneous but depends on the continuous stimulation of cells by serum factors: A labile protein inhibitor of transport, the effect of which is reversed by cAMP, is responsible for retention of c-Fos protein within the cytoplasm of serum-starved fibroblasts. In contrast, v-Fos proteins transduced by the murine retroviruses FBJ and FBR, which remain nuclear in the absence of serum, evade the translocation control, which therefore appears to contribute to their tumorigenic potential.

We studied c- fos gene expression in rat fibroblasts by microinjection of regulatory DNA sequences, such as the serum response element (SRE) present in c- fos promotor, in order to compete directly with such sequences for binding of putative regulatory factors. We show that an additional fos intragenic regulatory element (FIRE) is located at the end of exon 1. When coinjected with an SRE oligonucleotide, it induced c- fos expression in quiescent cells, whereas injection of SRE sequence alone failed to do so. Moreover, injection in quiescent cells of an SRE oligonucleotide together with a p- fos-lacZ construct containing the c- fos SRE as well as an in-frame insertion of FIRE resulted in a block to β-galactosidase expression that can be relieved by coinjection of the FIRE sequence.

Resume La proteine kinase decrite precedemment comme une activite endogene presente dans les particules ribonucleoproteiques contenant le RNA heterogene nucleaire dans les cellules HeLa ( Blanchard et al, Eur. J. Biochem. (1977) , 79 , 117–131) a ete partiellement purifiee par une combinaison de chromatographies sur DEAE cellulose, phosphocellulose et Sephacryl S-200. Elle est capable de phosphoryler des substrats exogenes parmi lesquels la caseine est le plus efficace. Ses proprietes ont ete trouvees tres semblables a celles decrites pour l'activite endogene. L'activite est independante de l'AMP cyclique ainsi que de la « calcium dependent regulator proteinet elle est inhibee par l'hemine. Son poids moleculaire a l'etat natif, determine par filtration sur gel, est de l'ordre de 48.000.

Small nuclear ribonucleoproteins (snRNPs) containing U1 and U5 snRNAs from HeLa cells have been fractionated using a combination of isopycnic centrifugation in cesium chloride and ion-exchange chromatography on DEAE-Sepharose. The procedure is based on the extreme stability conferred upon snRNPs by Mg2+ enabling them to withstand the very high ionic strength that prevails in cesium chloride. U1 snRNP prepared by this method contains all nine major proteins (68K, A, B, B′, C, D, E, F, G) corresponding to those previously identified by immunoprecipitation and is therefore precipitable by anti-RNP and anti-Sm antibodies. U5 snRNP purified in this way contains the common D to G proteins and is also enriched in a 25 × 103 Mr protein that may be U5 snRNP-specific. The core-resistant U5 snRNA sequence (nucleotide 84 to 3′ OH) covered by D to G proteins is extended by only six nucleotides. A similar situation is seen in U4-U6 snRNP, which we have obtained in a sufficiently pure form to examine protected sequences. However, the core-resistant sequence of U4 (nucleotide 116 to 3′ OH) in U4-U6 snRNP is extended by 37 nucleotides, suggesting that the protein composition of this particle could be more complex than that of U5 snRNP. The ribonucleoprotein organization of snRNPs is summarized and discussed in view of our current knowledge on snRNA sequences protected by proteins.