RsmY, a small regulatory RNA, is required in concert with RsmZ for GacA-dependent expression of biocontrol traits in Pseudomonas fluorescens CHA0.

In the plant-beneficial soil bacterium and biocontrol model organism Pseudomonas fluorescens CHA0, the GacS/GacA two-component system upregulates the production of biocontrol factors, i.e. antifungal secondary metabolites and extracellular enzymes, under conditions of slow, non-exponential growth. When activated, the GacS/GacA system promotes the transcription of a small regulatory RNA (RsmZ), which sequesters the small RNA-binding protein RsmA, a translational regulator of genes involved in biocontrol. The gene for a second GacA-regulated small RNA (RsmY) was detected in silico in various pseudomonads, and was cloned from strain CHA0. RsmY, like RsmZ, contains several characteristic GGA motifs. The rsmY gene was expressed in strain CHA0 as a 118 nt transcript which was most abundant in stationary phase, as revealed by Northern blot and transcriptional fusion analysis. Transcription of rsmY was enhanced by the addition of the strain's own supernatant extract containing a quorum-sensing signal and was abolished in gacS or gacA mutants. An rsmA mutation led to reduced rsmY expression, via a gacA-independent mechanism. Overexpression of rsmY restored the expression of target genes (hcnA, aprA) to gacS or gacA mutants. Whereas mutants deleted for either the rsmY or the rsmZ structural gene were not significantly altered in the synthesis of extracellular products (hydrogen cyanide, 2,4-diacetylphloroglucinol, exoprotease), an rsmY rsmZ double mutant was strongly impaired in this production and in its biocontrol properties in a cucumber-Pythium ultimum microcosm. Mobility shift assays demonstrated that multiple molecules of RsmA bound specifically to RsmY and RsmZ RNAs. In conclusion, two small, untranslated RNAs, RsmY and RsmZ, are key factors that relieve RsmA-mediated regulation of secondary metabolism and biocontrol traits in the GacS/GacA cascade of strain CHA0.

Developmental stage-specific expression of cyclic adenosine 3',5'-monophosphate response element-binding protein CREB during spermatogenesis involves alternative exon splicing.

Spermatogenesis is a temporally regulated developmental process by which the gonadotropin-responsive somatic Sertoli and Leydig cells act interdependently to direct the maturation of the germinal cells. The metabolism of Sertoli and Leydig cells is regulated by the pituitary gonadotropins FSH and LH, which, in turn, activate adenylate cyclase. Because the cAMP-second messenger pathway is activated by FSH and LH, we postulated that the cAMP-responsive element-binding protein (CREB) plays a physiological role in Sertoli and Leydig cells, respectively. Immunocytochemical analyses of rat testicular sections show a remarkably high expression of CREB in the haploid round spermatids and, to some extent, in pachytene spermatocytes and Sertoli cells. Although most of the CREB antigen is detected in the nuclei, some CREB antigen is also present in the cytoplasm. Remarkably, the cytoplasmic CREB results from the translation of a unique alternatively spliced transcript of the CREB gene that incorporates an exon containing multiple stop codons inserted immediately up-stream of the exons encoding the DNA-binding domain of CREB. Thus, the RNA containing the alternatively spliced exon encodes a truncated transcriptional transactivator protein lacking both the DNA-binding domain and nuclear translocation signal of CREB. Most of the CREB transcripts detected in the germinal cells contain the alternatively spliced exon, suggesting a function of the exon to modulate the synthesis of CREB. In the Sertoli cells we observed a striking cyclical (12-day periodicity) increase in the levels of CREB mRNA that coincides with the splicing out of the restrictive exon containing the stop codons. Because earlier studies established that FSH-stimulated cAMP levels in Sertoli cells are also cyclical, and the CREB gene promoter contains cAMP-responsive enhancers, we suggest that the alternative RNA splicing controls a positive autoregulation of CREB gene expression mediated by cAMP.

GENETIC AND BIOCHEMICAL ANALYSIS OF THE TESTIS-SPECIFIC ZINC FINGER PROTEIN CTCFL/BORIS

Abstract en FrançaisCTCFL a d'abord été identifié comme un paralogue de la protéine ubiquitaire CTCF en raison de sa forte homologie entre leurs onze « zinc fingers », un domaine de liaison à l'ADN. Parmi ses nombreux rôles, la liaison des zinc fingers de CTCF à la région de contrôle de l'empreinte (ICR) maternelle non-méthylée Igf2/H19, contrôle l'expression empreinte (monoallélique) de H19 et IGF2 dans les cellules somatiques. La méthylation de l'ICR Igf2/H19 paternelle est nécessaire à l'expression empreinte de ces deux gènes. Bien que le mécanisme par lequel l'ICR est méthylé soit mal compris, il est connu que l'établissement de la méthylation se produit pendant le développement des cellules germinales mâles et que les ADN méthyltransférases de novo DNMT3A et DNMT3L sont essentiels. Par conséquent, CTCFL fournit un bon candidat pour un rôle dans la méthylation de l'ICR paternelle Igf2/H19 en raison de son expression restreinte à certains types de cellules où la méthylation de l'ICR a lieu (spermatogonies et spermatocytes) ainsi qu'en raison sa capacité à lier les ICR lgf2/HÎ9 dans ces cellules. Les premiers travaux expérimentaux de cette thèse portent sur le rôle possible des mutations de CTCFL chez les patients atteints du syndrome de Silver-Russell (SRS), où une diminution de la méthylation de l'ICR IGF2/H19 a été observée chez 60% d'entre eux. Admettant que CTCFL pourrait être muté chez ces patients, j'ai examiné les mutations possibles de CTCFL chez 35 d'entre eux par séquençage de l'ADN et analyse du nombre de copies d'exons. N'ayant trouvé aucune mutation chez ces patients, cela suggère que les mutations de CTCFL ne sont pas associées au SRS. Les travaux expérimentaux suivants ont porté sur les modifications post-traductionnelles de CTCFL par la protéine SU MO « small ubiquitin-like modifier » (SUMO). La modification de protéines par SU MO change les interactions avec d'autres molécules (ADN ou protéines). Comme CTCFL régule sans doute l'expression d'un certain nombre de gènes dans le cancer et que plusieurs facteurs de transcription sont régulés par SUMO, j'ai mené des expériences pour déterminer si CTCFL est sumoylé. En effet, j'ai observé que CTCFL est sumoylated in vitro et in vivo et j'ai déterminé les deux résidus d'attachement de SUMO aux lysines 181 et 645. Utilisant les mutants de CTCFL K181R et K645R ne pouvant pas être sumoylated, j'ai évalué les conséquences fonctionnelles de la modification par SUMO. Je n'ai trouvé aucun changement significatif dans la localisation subcellulaire, la demi-vie ou la liaison à l'ADN, mais ai constaté que la sumoylation module à la fois {'activation CTCFL-dépendante et la répression de l'expression génique. Il s'agit de la première modification post-traductionnelle décrite pour CTCFL et les conséquences possibles de cette modification sont discutées pour le cancer et les testicules normaux. Avec cette thèse, j'espère avoir ajouté des résultats importants à l'étude de CTCFL et donné quelques idées pour de futures recherches.AbstractJeremiah Bernier-Latmani, Institute of Pathology, University of Lausanne, CHUVCTCFL was first identified as a paralog of the ubiquitous protein CTCF because of high homology between their respective eleven zinc fingers, a DNA binding domain. Among its many roles, CTCF zinc finger-mediated binding to the unmethylated maternal Igf2/H19 imprinting control region (ICR), controls the imprinted (monoallelic) expression of Igf2 and H19 in somatic cells. Methylation of the paternal Igf2/H19 ICR is necessary for the imprinted expression of the two genes. Although the mechanism by which the ICR is methylated is incompletely understood, it is known that establishment of methylation occurs during male germ cell development and the de novo DNA methyltransferases DNMT3A and DNMT3L are essential. Therefore, CTCFL provided a good candidate to play a role in methylation of the paternal Igf2/H19 ICR because of its restricted expression to cell types where ICR methylation takes place (spermatogonia and spermatocytes) and its ability to bind the Igf2/H19 ICR in these cells. The first experimental work of this thesis investigated the possible role of CTCFL mutations in Silver-Russell syndrome (SRS) patients, where it has been observed that 60% of the patients have reduced methylation of the IGF2/HÎ9 ICR. Reasoning that CTCFL could be mutated in these patients, I screened 35 patients for mutations in CTCFL by DNA sequencing and exon copy number analysis, I did not find any mutations in these patients suggesting that mutations of CTCFL are not associated with SRS. The next experimental work of my thesis focused on posttranslational modification of CTCFL by small ubiquitin-like modifier (SUMO) protein. SUMO modification of proteins changes the interactions with other molecules (DNA or protein). As CTCFL arguably regulates the expression of a number of genes in cancer and many transcription factors are regulated by SUMO, I conducted experiments to assess whether CTCFL is sumoylated. I found that CTCFL is sumoylated in vitro and in vivo and determined the two residues of SUMO attachment to be lysines 181 and 645. Using K181R, K645R mutated CTCFL- which cannot be detected to be sumoylated-1 assessed the functional consequences of SUMO modification. I found no significant changes in subcellular localization, half-life or DNA binding, but found that sumoylation modulates both CTCFL-dependent activation and repression of gene expression. This is the first posttranslational modification described for CTCFL and possible consequences of this modification are discussed in both cancer and normal testis. With this thesis, I hope I have added important findings to the study of CTCFL and provide some ideas for future research.

GacA-controlled activation of promoters for small RNA genes in Pseudomonas fluorescens.

The Gac/Rsm signal transduction pathway positively regulates secondary metabolism, production of extracellular enzymes, and biocontrol properties of Pseudomonas fluorescens CHA0 via the expression of three noncoding small RNAs, termed RsmX, RsmY, and RsmZ. The architecture and function of the rsmY and rsmZ promoters were studied in vivo. A conserved palindromic upstream activating sequence (UAS) was found to be necessary but not sufficient for rsmY and rsmZ expression and for activation by the response regulator GacA. A poorly conserved linker region located between the UAS and the -10 promoter sequence was also essential for GacA-dependent rsmY and rsmZ expression, suggesting a need for auxiliary transcription factors. One such factor involved in the activation of the rsmZ promoter was identified as the PsrA protein, previously recognized as an activator of the rpoS gene and a repressor of fatty acid degradation. Furthermore, the integration host factor (IHF) protein was found to bind with high affinity to the rsmZ promoter region in vitro, suggesting that DNA bending contributes to the regulated expression of rsmZ. In an rsmXYZ triple mutant, the expression of rsmY and rsmZ was elevated above that found in the wild type. This negative feedback loop appears to involve the translational regulators RsmA and RsmE, whose activity is antagonized by RsmXYZ, and several hypothetical DNA-binding proteins. This highly complex network controls the expression of the three small RNAs in response to cell physiology and cell population densities.

The SM protein Vps33 and the t-SNARE H(abc) domain promote fusion pore opening.

Intracellular membrane fusion proceeds via distinct stages of membrane docking, hemifusion and fusion pore opening and depends on interacting families of Rab, SNARE and SM proteins. Trans-SNARE complexes dock the membranes in close apposition. Efficient fusion requires further SNARE-associated proteins. They might increase the number of trans-SNARE complexes or the fusogenic potential of a single SNARE complex. We investigated the contributions of the SM protein Vps33 to hemifusion and pore opening between yeast vacuoles. Mutations in Vps33 that weaken its interactions with the SNARE complex allowed normal trans-SNARE pairing and lipid mixing but retarded content mixing. Deleting the H(abc) domain of the vacuolar t-SNARE Vam3, which interacts with Vps33, had the same effect. This suggests that SM proteins promote fusion pore opening by enhancing the fusogenic activity of a SNARE complex. They should thus be considered integral parts of the fusion machinery.

Nonstructural protein 3-4A: the Swiss army knife of hepatitis C virus.

Summary.  Hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) is a complex composed of NS3 and its cofactor NS4A. It harbours serine protease as well as NTPase/RNA helicase activities and is essential for viral polyprotein processing, RNA replication and virion formation. Specific inhibitors of the NS3-4A protease significantly improve sustained virological response rates in patients with chronic hepatitis C when combined with pegylated interferon-α and ribavirin. The NS3-4A protease can also target selected cellular proteins, thereby blocking innate immune pathways and modulating growth factor signalling. Hence, NS3-4A is not only an essential component of the viral replication complex and prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. This review provides a concise update on the biochemical and structural aspects of NS3-4A, its role in the pathogenesis of chronic hepatitis C and the clinical development of NS3-4A protease inhibitors.

Cis-trans interactions of cell surface receptors: biological roles and structural basis.

Cell surface receptors bind ligands expressed on other cells (in trans) in order to communicate with neighboring cells. However, an increasing number of cell surface receptors are found to also interact with ligands expressed on the same cell (in cis). These observations raise questions regarding the biological role of such cis interactions. Specifically, it is important to know whether cis and trans binding have distinct functional effects and, if so, how a single cell discriminates between interactions in cis versus trans. Further, what are the structural features that allow certain cell surface receptors to engage ligand both on the same as well as on an apposed cell membrane? Here, we summarize known examples of receptors that display cis-trans binding and discuss the emerging diversity of biological roles played by these unconventional two-way interactions, along with their structural basis.

FAM161A, associated with retinitis pigmentosa, is a component of the cilia-basal body complex and interacts with proteins involved in ciliopathies.

Retinitis pigmentosa (RP) is a retinal degenerative disease characterized by the progressive loss of photoreceptors. We have previously demonstrated that RP can be caused by recessive mutations in the human FAM161A gene, encoding a protein with unknown function that contains a conserved region shared only with a distant paralog, FAM161B. In this study, we show that FAM161A localizes at the base of the photoreceptor connecting cilium in human, mouse and rat. Furthermore, it is also present at the ciliary basal body in ciliated mammalian cells, both in native conditions and upon the expression of recombinant tagged proteins. Yeast two-hybrid analysis of binary interactions between FAM161A and an array of ciliary and ciliopathy-associated proteins reveals direct interaction with lebercilin, CEP290, OFD1 and SDCCAG8, all involved in hereditary retinal degeneration. These interactions are mediated by the C-terminal moiety of FAM161A, as demonstrated by pull-down experiments in cultured cell lines and in bovine retinal extracts. As other ciliary proteins, FAM161A can also interact with the microtubules and organize itself into microtubule-dependent intracellular networks. Moreover, small interfering RNA-mediated depletion of FAM161A transcripts in cultured cells causes the reduction in assembled primary cilia. Taken together, these data indicate that FAM161A-associated RP can be considered as a novel retinal ciliopathy and that its molecular pathogenesis may be related to other ciliopathies.

Small RNA as global regulator of carbon catabolite repression in Pseudomonas aeruginosa.

In the metabolically versatile bacterium Pseudomonas aeruginosa, the RNA-binding protein Crc is involved in catabolite repression of a range of degradative genes, such as amiE (encoding aliphatic amidase). We found that a CA-rich sequence (termed CA motif) in the amiE translation initiation region was important for Crc binding. The small RNA CrcZ (407 nt) containing 5 CA motifs was able to bind the Crc protein with high affinity and to remove it from amiE mRNA in vitro. Overexpression of crcZ relieved catabolite repression in vivo, whereas a crcZ mutation pleiotropically prevented the utilization of several carbon sources. The sigma factor RpoN and the CbrA/CbrB two-component system, which is known to maintain a healthy carbon-nitrogen balance, were necessary for crcZ expression. During growth on succinate, a preferred carbon source, CrcZ expression was low, resulting in catabolite repression of amiE and other genes under Crc control. By contrast, during growth on mannitol, a poor carbon source, elevated CrcZ levels correlated with relief of catabolite repression. During growth on glucose, an intermediate carbon source, CrcZ levels and amiE expression were intermediate between those observed in succinate and mannitol media. Thus, the CbrA-CbrB-CrcZ-Crc system allows the bacterium to adapt differentially to various carbon sources. This cascade also regulated the expression of the xylS (benR) gene, which encodes a transcriptional regulator involved in benzoate degradation, in an analogous way, confirming this cascade's global role.

A liver protein fraction regulating hormone-dependent in vitro transcription from the vitellogenin genes induces their expression in Xenopus oocytes.

Xenopus laevis oocytes were used to assay for trans-acting factors shown previously to be involved in the liver-specific regulation of the vitellogenin genes in vitro. To this end, crude liver nuclear extracts obtained from adult estrogen-induced Xenopus females were fractionated by heparin-Sepharose chromatography using successive elutions with 0.1, 0.35, 0.6, and 1.0 M KCl. When these four fractions were injected into oocytes, only the 0.6-M KCl protein fraction significantly stimulated mRNA synthesis from the endogenous B class vitellogenin genes. This same fraction induced estrogen-dependent in vitro transcription from the vitellogenin B1 promoter, suggesting that it contains at least a minimal set of basal transcription factors as well as two positive factors essential for vitellogenin in vitro transcription, i.e. the NF-I-like liver factor B and the estrogen receptor (ER). The presence of these two latter factors was determined by footprinting and gel retardation assays, respectively. In contrast, injection of an expression vector carrying the sequence encoding the ER was unable to activate transcription from the oocyte chromosomal vitellogenin genes. This suggests that the ER alone cannot overcome tissue-specific barriers and that one or several additional liver components participate in mediating tissue-specific expression of the vitellogenin genes. In this respect, we present evidence that the oocyte germinal vesicles contain an NF-I-like activity different from that found in hepatocytes of adult frogs. This observation might explain the lack of vitellogenin gene activation in oocytes injected with the ER cDNA only.

Changes of beta-amyloid precursor protein splice patterns in brain cell aggregate cultures.

The splice pattern of beta-amyloid precursor protein (beta-APP) has been studied in a variety of neuronal and glial cells and in brain cell aggregate cultures by the polymerase chain reaction (PCR). The brain-typical pattern, in which beta-APP695 is the dominant form, has been found only in aggregate cultures but not in any of the other cell types including neuronal cell lines. Selective elimination of glial cells from aggregates resulted in increased quantities of beta-APP695, whereas removal of neurons led to a reduction of beta-APP695 and to an elevation of beta-APP751 and beta-APP770. This shift of splice pattern was not observed in cocultures of the neuronal cell line PC 12 with primary astrocytes combined in a variety of cellular ratios. Blood serum, which is an essential component of these cultures, tested on aggregates, did not reduce the amount of beta-APP695 or have any marked effects on splice patterns generally. From these results it is concluded that investigations on brain-typical splicing of beta-APP require primary neurons. Neuronal cell lines may be no suitable model systems. Splicing events favoring production of beta-APP695 may mark an important, very early step of amyloid formation in the brain.

Immunodensity and mRNA expression of A2A adenosine, D2 dopamine, and CB1 cannabinoid receptors in postmortem frontal cortex of subjects with schizophrenia: effect of antipsychotic treatment.

RATIONALE: Dopamine D2 receptors are the main target of antipsychotic drugs. In the brain, D2 receptors coexpress with adenosine A2A and CB1 cannabinoid receptors, leading to functional interactions. OBJECTIVES: The protein and messenger RNA (mRNA) contents of A2A, D2, and CB1 receptors were quantified in postmortem prefrontal cortex of subjects with schizophrenia. MATERIALS AND METHODS: The study was performed in subjects suffering schizophrenia (n=31) who mainly died by suicide, matched with non-schizophrenia suicide victims (n=13) and non-suicide controls (n=33). The density of receptor proteins was evaluated by immunodetection techniques, and their relative mRNA expression was quantified by quantitative real-time polymerase chain reaction. RESULTS: In schizophrenia, the densities of A2A (90+/-6%, n=24) and D2-like receptors (95+/-5%, n=22) did not differ from those in controls (100%). Antipsychotic treatment did not induce changes in the protein expression. In contrast, the immunodensity of CB1 receptors was significantly decreased (71+/-7%, n=11; p<0.05) in antipsychotic-treated subjects with schizophrenia but not in drug-free subjects (104+/-13%, n=11). The relative mRNA amounts encoding for A2A, D2, and CB1 receptors were similar in brains of drug-free, antipsychotic-treated subjects with schizophrenia and controls. CONCLUSIONS: The findings suggest that antipsychotics induce down-regulation of CB1 receptors in brain. Since A2A, D2, and CB1 receptors coexpress on brain GABAergic neurons and reductions in markers of GABA neurotransmission have been identified in schizophrenia, a lower density of CB1 receptor induced by antipsychotics could represent an adaptative mechanism that reduces the endocannabinoid-mediated suppression of GABA release, contributing to the normalization of cognitive functions in the disorder.

Ceftriaxone--bilirubin-albumin interactions in the neonate: an in vivo study.

The in vivo bilirubin-albumin binding interaction of ceftriaxone (CRO) was investigated in 14 non-jaundiced newborns, aged 33-42 weeks of gestation, during the first few days of life after they had reached stable clinical condition. CRO (50 mg/kg) was infused intravenously over 30 min. The competitive binding effect of CRO on the bilirubin-albumin complex was estimated by determining the reserve albumin concentration (RAC) at baseline, at the end of CRO infusion, and at 15 and 60 min thereafter. Immediately after the end of drug administration, RAC decreased from 91.9 (+/- 25.1) mumol/l to 38.6 (+/- 10.1) mumol/l (P = 0.0001). At the same time the plasma bilirubin toxicity index (PBTI) increased from 0.64 (+/- 0.40) before drug infusion to 0.96 (+/- 0.44) thereafter (P = 0.0001). The highest displacement factor (DF) was calculated to be 2.8 (+/- 0.6) at the end of drug infusion. Average total serum bilirubin concentrations decreased from a baseline value of 59.6 (+/- 27.0) mumol/l to 55.2 (+/- 27.1) mumol/l (P = 0.026). Sixty minutes after the end of CRO infusion, RAC was 58.3 (+/- 21.7) mumol/l, PBTI regained baseline, but DF was still 1.9 (+/- 0.2). No adverse events were recorded. Our results demonstrate significant competitive interaction of CRO with bilirubin-albumin binding in vivo. Thus, ceftriaxone should not be given to the neonate at risk of developing bilirubin encephalopathy.

Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0, an antagonist of root-pathogenic fungi, the GacS/GacA two-component system tightly controls the expression of antifungal secondary metabolites and exoenzymes at a posttranscriptional level, involving the RNA-binding protein and global regulator of secondary metabolism RsmA. This protein was purified from P. fluorescens, and RNA bound to it was converted to cDNA, which served as a probe to isolate the corresponding chromosomal locus, rsmZ. This gene encoded a regulatory RNA of 127 nucleotides and a truncated form lacking 35 nucleotides at the 3' end. Expression of rsmZ depended on GacA, increased with increasing population density, and was stimulated by the addition of a solvent-extractable extracellular signal produced by strain CHA0 at the end of exponential growth. This signal appeared to be unrelated to N-acyl-homoserine lactones. A conserved upstream element in the rsmZ promoter, but not the stress sigma factor RpoS, was involved in rsmZ expression. Overexpression of rsmZ effectively suppressed the negative effect of gacS and gacA mutations on target genes, i.e., hcnA (for hydrogen cyanide synthase) and aprA (for the major exoprotease). Mutational inactivation of rsmZ resulted in reduced expression of these target genes in the presence of added signal. Overexpression of rsmA had a similar, albeit stronger negative effect. These results support a model in which GacA upregulates the expression of regulatory RNAs, such as RsmZ of strain CHA0, in response to a bacterial signal. By a titration effect, RsmZ may then alleviate the repressing activity of RsmA on the expression of target mRNAs.

C/EBPbeta couples dopamine signalling to substance P precursor gene expression in striatal neurones.

Dopamine-induced changes in striatal gene expression are thought to play an important role in drug addiction and compulsive behaviour. In this study we report that dopamine induces the expression of the transcription factor CCAAT/Enhancer Binding Protein beta (C/EBP)-beta in primary cultures of striatal neurones. We identified the preprotachykinin-A (PPT-A) gene coding for substance P and neurokinin-A as a potential target gene of C/EBPbeta. We demonstrated that C/EBPbeta physically interacts with an element of the PPT-A promoter, thereby facilitating substance P precursor gene transcription. The regulation of PPT-A gene by C/EBPbeta could subserve many important physiological processes involving substance P, such as nociception, neurogenic inflammation and addiction. Given that substance P is known to increase dopamine signalling in the striatum and, in turn, dopamine increases substance P expression in medium spiny neurones, our results implicate C/EBPbeta in a positive feedback loop, changes of which might contribute to the development of drug addiction.