Involvement of the RNA-binding protein ARE/poly(U)-binding factor 1 (AUF1) in the cytotoxic effects of proinflammatory cytokines on pancreatic beta cells.

AIMS/HYPOTHESIS: Chronic exposure of pancreatic beta cells to proinflammatory cytokines leads to impaired insulin secretion and apoptosis. ARE/poly(U)-binding factor 1 (AUF1) belongs to a protein family that controls mRNA stability and translation by associating with adenosine- and uridine-rich regions of target messengers. We investigated the involvement of AUF1 in cytokine-induced beta cell dysfunction. METHODS: Production and subcellular distribution of AUF1 isoforms were analysed by western blotting. To test for their role in the control of beta cell functions, each isoform was overproduced individually in insulin-secreting cells. The contribution to cytokine-mediated beta cell dysfunction was evaluated by preventing the production of AUF1 isoforms by RNA interference. The effect of AUF1 on the production of potential targets was assessed by western blotting. RESULTS: MIN6 cells and human pancreatic islets were found to produce four AUF1 isoforms (p42>p45>p37>p40). AUF1 isoforms were mainly localised in the nucleus but were partially translocated to the cytoplasm upon exposure of beta cells to cytokines and activation of the ERK pathway. Overproduction of AUF1 did not affect glucose-induced insulin secretion but promoted apoptosis. This effect was associated with a decrease in the production of the anti-apoptotic proteins, B cell leukaemia/lymphoma 2 (BCL2) and myeloid cell leukaemia sequence 1 (MCL1). Silencing of AUF1 isoforms restored the levels of the anti-apoptotic proteins, attenuated the activation of the nuclear factor-κB (NFκB) pathway, and protected the beta cells from cytokine-induced apoptosis. CONCLUSIONS/INTERPRETATION: Our findings point to a contribution of AUF1 to the deleterious effects of cytokines on beta cell functions and suggest a role for this RNA-binding protein in the early phases of type 1 diabetes.

Transcriptional regulation of RNA polymerase III in mammalian cells

L'ARN Polymérase III (Pol III) transcrit un ensemble de petits ARN non traduits impliqués dans des processus cellulaires tels que la biosynthèse des protéines, la maturation des ARNs ou le contrôle transcriptionnel. De ce fait, la Pol III joue un rôle important dans la régulation de la croissance et la prolifération cellulaire. L'initiation de la transcription par la Pol III nécessite l'interaction entre des facteurs de transcription et le complexe de la Pol III lui-même. Un sous- complexe de la Pol III, composé de 3 sous-unités, HsRPC3, HsRPC6 et HsRPC7 sert d'intermédiaire dans cette interaction. Dans cette étude, nous avons caractérisé une nouvelle sous-unité de la Pol III, HsRPC7-Like, homologue à HsRPC7. Nous avons montré que ces deux homologues se trouvent spécifiquement chez les vertébrés. Ils proviennent d'un ancêtre commun qui, après duplication il y a 600 millions d'années, a donné naissance à ces deux paralogues. Dans les cellules humaines, deux formes de Pol III coexistent : l'une contientt HsRPC7, l'autre HsRPC7-Like. Nous avons localisé, à l'échelle du génome entier, la présence de ces deux formes de Pol III dans des cellules humaines et dans le foie de souris. Les deux sous-unités ont démontré des caractéristiques identiques, suggérant qu'elles possèdent des fonctions similaires. Cependant, nous avons analysé les motifs d'expression des gènes codant pour RPC7 et RPC7-Like dans des lignées cellulaires dans des conditions variées telles que la concentration de sérum et la densité cellulaire, ainsi que les motifs d'expression dans le foie de souris et des cellules d'hépatocarcinome de souris. Nos résultats suggèrent que l'expression de ces deux sous-untiés varie en fonction de l'activité de prolifération de la cellule. - RNA polymerase III (Pol III) transcribes a set of genes coding for short untranslated RNAs involved in essential cellular processes as for example protein biosynthesis, RNA maturation, and transcriptional control. Thereby Pol III plays an important role in regulating cell growth and proliferation. Initiation of Pol III transcription requires interactions between transcription factors and the Pol III core complex. A Pol III sub-complex composed of three subunits, HsRPC3, HsRPC6, and HsRPC7 mediates this interaction. In this study, we have characterized a new Pol III subunit, HsRPC7-Like, an homologue of HsRPC7. We have shown that these two homologues are specific to vertebrates and originate from an ancestor gene that duplicated 600 mio years ago to give birth to two paralogues. In human cells, two forms of Pol III coexist, one containing HsRPC7 and the other HsRPC7-Like. We have localized, genome-wide, these two Pol III forms in human cells and mouse liver. Both subunits were found on all types of Pol III genes, suggesting that they share similar function. However, we analysed the expression patterns of the RPC7 and RPC7-Like coding genes under various conditions of serum concentration and cell density in different cell lines, as well as expression patterns in mouse liver and mouse hepatocarcinoma cells. Our results suggest that the expression of these two subunits varies with the proliferation rate of the cell.

CD30 in PTCLS: correlation between RNA and protein levels in a large european series from the LYSA

Introduction: CD22 is expressed on most B-non-Hodgkin lymphomas (NHL); inotuzumab ozogamicin (INO) is an anti-CD22 antibody conjugated to calicheamicin. This study evaluated the safety and tolerability of INO plus R-CVP in patients (pts) with relapsed/refractory CD22+ B-NHL. Efficacy data were also collected. Methods: Part 1 of this open-label study identified a maximum tolerated dose (MTD) of INO 0.8mg/m,2 on day 2 plus R-CVP (rituximab 375mg/m,2 cyclophosphamide 750mg/m,2 and vincristine 1.4mg/m,2 on day 1; prednisone 40mg/m,2 on days 1-5) every 21 days. Subsequently, pts were enrolled in the MTD confirmation cohort (part 2, n = 10), which required a dose-limiting toxicity rate of <33% in cycle 1 and <4 pts discontinuing prior to cycle 3 due to an adverse event (AE) in the MTD expansion cohort (part 3, n = 22), which explored preliminary activity. Results: Parts 2 and 3 enrolled 32 pts: 16 pts with diffuse large B-cell lymphoma, 15 with follicular lymphoma and one with mantle cell lymphoma. Median age was 64.5 years (range 44-81 years); 34% of pts had 1 prior regimen, 34% had 2, 28% had ≥3 and 3% had none (median 2; range 0-6).Median treatment duration was five cycles (range 1-6). Part 2 confirmed the MTD as standard dose R-CVP plus INO 0.8mg/m,2; 2/10 pts had a dose-limiting toxicity (grade 3 increased ALT/AST, grade 4 neutropenia requiring G-CSF). One pt discontinued because of an AE prior to cycle 3. Common treatment-related AEs were thrombocytopenia (78%), neutropenia (66%), fatigue (50%), leukopenia (50%), nausea (41%) and lymphopenia (38%); common grade 3/4 AEs were neutropenia (63%), thrombocytopenia (53%), leukopenia (38%) and lymphopenia (31%). There was one case of treatment-related fatal pneumonia with grade 4 neutropenia. Ten pts discontinued treatment due to AEs; thrombocytopenia/delayed platelet recovery was the leading cause (grade 1/2, n = 6; grade 3/4, n = 3). Objective response rate (ORR) was 77% (n = 24/31 evaluable pts), including 26% (n=8/31) with complete response (CR); three pts had stable disease. Of the pts with follicular lymphoma, ORR was 100% (n = 15/15), including seven pts with CR. Of the pts with diffuse large B-cell lymphoma, ORR was 60% (n = 9/16), including one pt with CR. Conclusions: Results suggest that INOplus R-CVP has acceptable toxicity and promising activity in relapsed/refractory CD22+ B-NHL. The most common grade 3/4 AEs were hematologic. Follow-up for progression-free and overall survival is ongoing.

The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA

The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3'UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT-qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.

The overexpression of the trypanosomatid-exclusive TcRBP19 RNA-binding protein affects cellular infection by Trypanosoma cruzi

To characterise the trypanosomatid-exclusive RNA-binding protein TcRBP19, we analysed the phenotypic changes caused by its overexpression. Although no evident changes were observed when TcRBP19 was ectopically expressed in epimastigotes, the metacyclogenesis process was affected. Notably, TcRBP19 overexpression also led to a decrease in the number of infected mammalian cells. These findings suggest that TcRBP19 may be involved in the life cycle progression of the Trypanosoma cruzi parasite.

Recombinant hepatitis C virus-envelope protein 2 interactions with low-density lipoprotein/CD81 receptors

Hepatitis C virus (HCV) envelope protein 2 (E2) is involved in viral binding to host cells. The aim of this work was to produce recombinant E2B and E2Y HCV proteins in Escherichia coli and Pichia pastoris, respectively, and to study their interactions with low-density lipoprotein receptor (LDLr) and CD81 in human umbilical vein endothelial cells (HUVEC) and the ECV304 bladder carcinoma cell line. To investigate the effects of human LDL and differences in protein structure (glycosylated or not) on binding efficiency, the recombinant proteins were either associated or not associated with lipoproteins before being assayed. The immunoreactivity of the recombinant proteins was analysed using pooled serum samples that were either positive or negative for hepatitis C. The cells were immunophenotyped by LDLr and CD81 using flow cytometry. Binding and binding inhibition assays were performed in the presence of LDL, foetal bovine serum (FCS) and specific antibodies. The results revealed that binding was reduced in the absence of FCS, but that the addition of human LDL rescued and increased binding capacity. In HUVEC cells, the use of antibodies to block LDLr led to a significant reduction in the binding of E2B and E2Y. CD81 antibodies did not affect E2B and E2Y binding. In ECV304 cells, blocking LDLr and CD81 produced similar effects, but they were not as marked as those that were observed in HUVEC cells. In conclusion, recombinant HCV E2 is dependent on LDL for its ability to bind to LDLr in HUVEC and ECV304 cells. These findings are relevant because E2 acts to anchor HCV to host cells; therefore, high blood levels of LDL could enhance viral infectivity in chronic hepatitis C patients.

Mitotic functions for SNAP45, a subunit of the small nuclear RNA-activating protein complex SNAPc.

The small nuclear RNA-activating protein complex SNAP(c) is required for transcription of small nuclear RNA genes and binds to a proximal sequence element in their promoters. SNAP(c) contains five types of subunits stably associated with each other. Here we show that one of these polypeptides, SNAP45, also known as PTF delta, localizes to centrosomes during parts of mitosis, as well as to the spindle midzone during anaphase and the mid-body during telophase. Consistent with localization to these mitotic structures, both down- and up-regulation of SNAP45 lead to a G(2)/M arrest with cells displaying abnormal mitotic structures. In contrast, down-regulation of SNAP190, another SNAP(c) subunit, leads to an accumulation of cells with a G(0)/G(1) DNA content. These results are consistent with the proposal that SNAP45 plays two roles in the cell, one as a subunit of the transcription factor SNAP(c) and another as a factor required for proper mitotic progression.

A short motif in Drosophila SECIS Binding Protein 2 provides differential binding affinity to SECIS RNA hairpins

Selenoproteins contain the amino acid selenocysteine which is encoded by a UGA Sec codon. Recoding UGA Sec requires a complex mechanism, comprising the cis-acting SECIS RNA hairpin in the 3′UTR of selenoprotein mRNAs, and trans-acting factors. Among these, the SECIS Binding Protein 2 (SBP2) is central to the mechanism. SBP2 has been so far functionally characterized only in rats and humans. In this work, we report the characterization of the Drosophila melanogaster SBP2 (dSBP2). Despite its shorter length, it retained the same selenoprotein synthesis-promoting capabilities as the mammalian counterpart. However, a major difference resides in the SECIS recognition pattern: while human SBP2 (hSBP2) binds the distinct form 1 and 2 SECIS RNAs with similar affinities, dSBP2 exhibits high affinity toward form 2 only. In addition, we report the identification of a K (lysine)-rich domain in all SBP2s, essential for SECIS and 60S ribosomal subunit binding, differing from the well-characterized L7Ae RNA-binding domain. Swapping only five amino acids between dSBP2 and hSBP2 in the K-rich domain conferred reversed SECIS-binding properties to the proteins, thus unveiling an important sequence for form 1 binding.

Tumor necrosis factor and transforming growth factor β regulate clock genes by controlling the expression of the cold inducible RNA-binding protein (CIRBP).

The circadian clock drives the rhythmic expression of a broad array of genes that orchestrate metabolism, sleep wake behavior, and the immune response. Clock genes are transcriptional regulators engaged in the generation of circadian rhythms. The cold inducible RNA-binding protein (CIRBP) guarantees high amplitude expression of clock. The cytokines TNF and TGFβ impair the expression of clock genes, namely the period genes and the proline- and acidic amino acid-rich basic leucine zipper (PAR-bZip) clock-controlled genes. Here, we show that TNF and TGFβ impair the expression of Cirbp in fibroblasts and neuronal cells. IL-1β, IL-6, IFNα, and IFNγ do not exert such effects. Depletion of Cirbp is found to increase the susceptibility of cells to the TNF-mediated inhibition of high amplitude expression of clock genes and modulates the TNF-induced cytokine response. Our findings reveal a new mechanism of cytokine-regulated expression of clock genes.

Interactions of Adeno-associated virus Rep78 protein with the host cell

Abstract : Adeno-associated virus (AAV) is a small DNA virus belonging to the familiy of Parvoviridae. Its genome contains two genes : the rep gene encoding four non structural proteins (Rep78, 68, 52 and 40) implicated in transcription, replication and site-specific integration of the viral DNA and the cap gene encoding three capsid proteins. AAV does not cause any disease, but is studied in view of its potential use to treat several diseases. An interesting property of AAV is its antiproliferative effect. Two elements of AAV can inhibit cell growth. Firstly, the single stranded viral DNA is recognized in cells as damaged DNA leading to either a G2 block or cell death depending on p53 status. Secondly, the two larger Rep proteins (Rep78 and 68) also arrest the cell cycle when they are expressed at high levels. Rep78 in particular induces a complete cell cycle arrest in all the phases, including S phase. Such a strong S phase arrest is rarely seen in other conditions. It was thus interesting to determine how Rep78 could induce it. We found that this strong block is the consequence of Rep78's effects on at least two pathways. Rep78 induces a DNA damage response by producing nicks in the cellular chromatin. Furthermore, Rep78 can bind to the cellular phosphatase Cdc25A and prevent its binding to its substrates CDK2 and CDK1, thus inhibiting its activity. A mutational analysis of Rep78 protein determined that its endonuclease activity is responsible for the DNA damage response and its zinc finger domain for Cdc25A inhibition. The combined expression of two mutants each defective for one of these activities, or these two activities obtained independently of Rep78, could restore the complete cell cycle block, indicating that these two effects of Rep78 are likely to explain completely the cell cycle block it induces. Secondly, the lack of pathogenicity of AAV, its broad range of infection and its ability to integrate site-specifically in human chromosome 19 make it an interesting potential vector for gene therapy. However site-specific integration is only possible in the presence of Rep78/68 whose gene is removed in recombinant AAV vectors. In this part of the study, we tried to introduce Rep protein separately from recombinant AAV vectors to promote their site-specific integration. For that purpose, a fusion protein, TAT-Rep, comprising Rep78/68 joined to the human immunodeficiency virus Tat protein was produced. It had the ability to enter cells and remain active there for a short period. Its activity was sufficient to mediate transcription from the p5 promoter, second-strand synthesis of a recombinant AAV and probably site-specific integration. Résumé : Le virus associé à l'adénovirus (AAV) est un petit virus à ADN qui fait partie de la famille des Parvoviridae. Son génome contient deux gènes : le gène rep code pour quatre protéines (Rep78, 68, 52 et 40) qui participent à la transcription, la réplication et l'intégration du virus et le gène cap code pour les trois protéines de capside. AAV ne produit pas de maladie, mais pourrait au contraire être utilisé pour en soigner. Sa bénignité, sa capacité à infecter différents types de cellules et son intégration spécifique en font un vecteur potentiel pour la thérapie génique. Pour qu'il puisse s'intégrer spécifiquement, il a besoin de la protéine Rep78 ou 68, mais ce gène doit être enlevé des vecteurs pour la thérapie génique. Le but de la première partie de cette étude était d'introduire Rep78 ou 68 dans des cellules en même temps qu'un AAV recombinant, mais indépendamment afin de permettre une intégration spécifique. La stratégie utilisée était de produire une protéine de fusion (TAT-Rep) qui peut entrer dans des cellules si elle est présente dans leur milieu. Cette protéine entrait bien dans les cellules et y était active favorisant ainsi l'intégration spécifique. Une deuxième propriété d'AAV, son effet anti-prolifératif, est intéressante dans le cadre de certaines maladies comme le cancer. Deux éléments d'AAV en sont responsables. D'abord, son ADN simple brin active une réponse cellulaire à l'ADN endommagé et arrête les cellules en G2 ou provoque leur mort. De plus, la protéine Rep78 d'AAV peut fortement bloquer le cycle cellulaire à toutes les phases, même en phase S, ce qui est rare. C'est pourquoi nous avons essayé de comprendre cet effet. Nous avons remarqué que Rep78 doit agir sur deux fronts pour obtenir ce fort bloc. D'un côté, Rep78 introduit des coupures simple brin sur l'ADN de la cellule ce qui active une réponse cellulaire à l'ADN endommagé qui passe par ATM. D'un autre côté, Rep78 lie une phosphatase cellulaire, Cdc25A, et l'empêche ainsi de lier ses substrats CDK2 et CDK1 et donc d'être active. Finalement, à l'aide de mutants de Rep78, nous avons déterminé que l'activité endonuclease de Rep78 était nécessaire pour induire une réponse cellulaire via ATM et que le domaine C-terminal appelé «zinc finger » était responsable de la liaison avec Cdc25A. En co-exprimant deux mutants, qui n'ont chacun qu'un des effets de Rep78, ou en obtenant les deux effets de Rep78 indépendamment d'elle, nous avons obtenu un bloc complet du cycle cellulaire similaire à celui obtenu avec Rep78. Il est donc probable que ces deux effets de Rep78 sont suffisants pour expliquer comment elle arrive à arrêter le cycle cellulaire si efficacement.

RNA pentaloop structures as effective targets of regulators belonging to the RsmA/CsrA protein family.

In the Gac/Rsm signal transduction pathway of Pseudomonas fluorescens CHA0, the dimeric RNA-binding proteins RsmA and RsmE, which belong to the vast bacterial RsmA/CsrA family, effectively repress translation of target mRNAs containing a typical recognition sequence near the translation start site. Three small RNAs (RsmX, RsmY, RsmZ) with clustered recognition sequences can sequester RsmA and RsmE and thereby relieve translational repression. According to a previously established structural model, the RsmE protein makes optimal contacts with an RNA sequence 5'- (A)/(U)CANGGANG(U)/(A)-3', in which the central ribonucleotides form a hexaloop. Here, we questioned the relevance of the hexaloop structure in target RNAs. We found that two predicted pentaloop structures, AGGGA (in pltA mRNA encoding a pyoluteorin biosynthetic enzyme) and AAGGA (in mutated pltA mRNA), allowed effective interaction with the RsmE protein in vivo. By contrast, ACGGA and AUGGA were poor targets. Isothermal titration calorimetry measurements confirmed the strong binding of RsmE to the AGGGA pentaloop structure in an RNA oligomer. Modeling studies highlighted the crucial role of the second ribonucleotide in the loop structure. In conclusion, a refined structural model of RsmE-RNA interaction accommodates certain pentaloop RNAs among the preferred hexaloop RNAs.

Posttranscriptional repression of GacS/GacA-controlled genes by the RNA-binding protein RsmE acting together with RsmA in the biocontrol strain Pseudomonas fluorescens CHA0.

In the plant-beneficial soil bacterium Pseudomonas fluorescens CHA0, the production of biocontrol factors (antifungal secondary metabolites and exoenzymes) is controlled at a posttranscriptional level by the GacS/GacA signal transduction pathway involving RNA-binding protein RsmA as a key regulatory element. This protein is assumed to bind to the ribosome-binding site of target mRNAs and to block their translation. RsmA-mediated repression is relieved at the end of exponential growth by two GacS/GacA-controlled regulatory RNAs RsmY and RsmZ, which bind and sequester the RsmA protein. A gene (rsmE) encoding a 64-amino-acid RsmA homolog was identified and characterized in strain CHA0. Overexpression of rsmE strongly reduced the expression of target genes (hcnA, for a hydrogen cyanide synthase subunit; aprA, for the main exoprotease; and phlA, for a component of 2,4-diacetylphloroglucinol biosynthesis). Single null mutations in either rsmA or rsmE resulted in a slight increase in the expression of hcnA, aprA, and phlA. By contrast, an rsmA rsmE double mutation led to strongly increased and advanced expression of these target genes and completely suppressed a gacS mutation. Both the RsmE and RsmA levels increased with increasing cell population densities in strain CHA0; however, the amount of RsmA showed less variability during growth. Expression of rsmE was controlled positively by GacA and negatively by RsmA and RsmE. Mobility shift assays demonstrated specific binding of RsmE to RsmY and RsmZ RNAs. The transcription and stability of both regulatory RNAs were strongly reduced in the rsmA rsmE double mutant. In conclusion, RsmA and RsmE together account for maximal repression in the GacS/GacA cascade of strain CHA0.

Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) Protein: Localization in Endocytic-coated Pits, Interactions with Clathrin, and the Impact of Overexpression on Clathrin-mediated Traffic

The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure-function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP-CALM was targeted to the plasma membrane-coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP-CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP-CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin-CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.

Effects of and interactions between the extent of silage fermentation and protein and protein supplementation in lactating dairy cows

Selostus: Säilörehun käymisasteen ja valkuaistäydennyksen vaikutus maidon tuotantoon