The role of AUF1 in AU-rich interleukin-6 mRNA regulation

Abstract: The AU-rich elements (AREs) consisting of repeated AUUUA motifs confer rapid degradation to many cellular mRNAs when present in the 3' untranslated region (3'UTR). We have studied the instability of interleukin-6 mRNA by grafting its 3' untranslated region to a stable green fluorescent protein mRNA. Subsequent scanning mutagenesis identified two conserved elements, which taken together account for most of the instability. The first corresponds to a short non-canonical AU-rich element. The other comprises a sequence predicted to form astern-loop structure. Both elements need to be present in order to confer full instability (Paschoud et al. 2006). Destabilization of ARE-containing mRNAs is thought to involve ARE-binding proteins such as AUF1. We tested whether AUF1 binding to interleukin-6 mRNA correlates with decreased mRNA stability. Overexpression of myc-tagged p37AUFl and p42AUF1 as well as suppression of all four AUF1 isoforms by RNA interference stabilized the interleukin-6 mRNA. Furthermore, the interleukin-6 mRNA co-immunoprecipitated specifically with myc-tagged p37AUF1 and p42AUF1 in cell extracts. Both the stabilization and AUF1-binding required the non-canonical AU-rich sequence. These results indicate that AUF1 binds to the AU-rich element in vivo and promotes interleukin6 mRNA degradation. The combination of mRNA co-immunoprecipitation with microarray technology revealed that at least 500 cellular mRNAs associate with AUF1. Résumé: "La présence d'éléments riches en A et U (ARE), en particulier les motifs répétés d'AUUUA dans la région 3' non traduite, confère une dégradation rapide à beaucoup d'ARN cellulaires. Nous avons étudié l'instabilité de l'ARN codant pour l'interleukine 6 en greffant sa région 3' non traduite à un ARN stable codant pour la protéine fluorescente verte. La mutagenèse systématique des séquences non traduites a permis l'identification de deux éléments conservés qui confèrent l'instabilité à l'ARN. Le premier correspond à un élément AU-riche non canonique court. Le second comporte une structure en 'épingle à cheveux'. Tous les deux éléments doivent être présents afin de conférer une instabilité complète (Paschoud et al. 2006). On pense que des protéines telles que AUF1, pouvant se lier aux éléments ARE, sont impliquées dans la dégradation des ARN messagers. Nous avons examiné si la liaison de AUFl sur l'ARN de l'interleukine 6 corrèle avec une stabilité diminuée. La surexpression des protéines p37AUF1 et de p42AUF1 myc-étiquetées ainsi que la suppression de chacun des quatre isoformes de AUF1 par interférence d'ARN a stabilisé l'ARN messager d'interleukine 6. En outre, cet ARN co-immunoprécipite spécifiquement avec p37AUF1 et p42AUF1 dans des extraits cellulaires. La présence de l'élément AUriche non canonique est nécessaire pour la stabilisation de l'ARN et sa liaison avec AUFI. Ces résultats indiquent qu'AUF1 se lie à l'élément AU-riche in vivo et favorise la dégradation de l'ARN messager d'interleukine 6. La combinaison des techniques de coimmunoprécipitation des ARN messagers et des analyses par `microarray' indique qu'au moins 500 ARN cellulaires s'associent à AUF1.

Cloning, characterization and regulation of the soluble guanylyl cyclase alpha1 and beta1 genes

Cell signaling by nitric oxide (NO) through soluble guanylyl cyclase (sGC) and cGMP production regulates physiological responses such as smooth muscle relaxation, neurotransmission, and cell growth and differentiation. Although the NO receptor, sGC, has been studied extensively at the protein level, information on regulation of the sGC genes remains elusive. In order to understand the molecular mechanisms involved at the level of gene expression, cDNA and genomic fragments of the murine sGCα1 subunit gene were obtained through library screenings. Using the acquired clones, the sGCα 1 gene structure was determined following primer extension, 3 ′RACE and intron/exon boundary analyses. The basal activity of several 5′-flanking regions (putative promoter regions) for both the α1 and β1 sGC subunits were determined following their transfection into mouse N1E-115 neuroblastoma and rat RENE1Δ14 uterine epithelial cells using a luciferase reporter plasmid. Using the sGC sequences, real-time RT-PCR assays were designed to measure mRNA levels of the sGC α1 and β1 genes in rat, mouse and human. Subsequent studies found that uterine sGC mRNA and protein levels decreased rapidly in response to 17β-estradiol (estrogen) in an in vivo rat model. As early as 1 hour following treatment, mRNA levels of both sGC mRNAs decreased, and reached their lowest level of expression after 3 hours. This in vivo response was completely blocked by the pure estrogen receptor antagonist, ICI 182,780, was not seen in several other tissues examined, did not occur in response to other steroid hormones, and was due to a post-transcriptional mechanism. Additional studies ex vivo and in various cell culture models suggested that the estrogen-mediated decreased sGC mRNA expression did not require signals from other tissues, but may require cell communication or paracrine factors between different cell types within the uterus. Using chemical inhibitors and molecular targeting in other related studies, it was revealed that c-Jun-N-terminal kinase (JNK) signaling was responsible for decreased sGC mRNA expression in rat PC12 and RFL-6 cells, two models previously determined to exhibit rapid decreased sGC mRNA expression in response to different stimuli. To further investigate the post-transcriptional gene regulation, the full length sGCα1 3′-untranslated region (3′UTR) was cloned from rat uterine tissue and ligated downstream of the rabbit β-globin gene and expressed as a chimeric mRNA in the rat PC12 and RFL-6 cell models. Expression studies with the chimeric mRNA showed that the sGCα 1 3′UTR was not sufficient to mediate the post-transcriptional regulation of its mRNA by JNK or cAMP signaling in PC12 and RFL-6 cells. This study has provided numerous valuable tools for future studies involving the molecular regulation of the sGC genes. Importantly, the present results identified a novel paradigm and a previously unknown signaling pathway for sGC mRNA regulation that could potentially be exploited to treat diseases such as uterine cancers, neuronal disorders, hypertension or various inflammatory conditions. ^

Trypanosoma cruzi-cardiomyocytes: new contributions regarding a better understanding of this interaction

The present paper summarizes new approaches regarding the progress done to the understanding of the interaction of Trypanosoma cruzi-cardiomyocytes. Mannose receptors localized at the surface of heart muscle cell are involved in binding and uptake of the parasite. One of the most striking events in the parasite-heart muscle cells interaction is the disruption of the actin cytoskeleton. We have investigated the regulation of the actin mRNA during the cytopathology induced in myocardial cells by the parasite. T. cruzi invasion increases calcium resting levels in cardiomyocytes. We have previously shown that Ca2+ ATPase of the sarcoplasmic reticulum (SERCA) is involved in the invasion of T. cruzi in cardiomyocytes. Treating the cells with thapsigargin, a drug that binds to all SERCA ATPases and causes depletion of intracellular calcium stores, we found a 75% inhibition in the T. cruzi-cardiomyocytes invasion.

Glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR): cellular localization, lesion-affected expression, and impaired regenerative axonal growth.

Glucose-dependent insulinotropic polypeptide (GIP) was initially described to be rapidly regulated by endocrine cells in response to nutrient ingestion, with stimulatory effects on insulin synthesis and release. Previously, we demonstrated a significant up-regulation of GIP mRNA in the rat subiculum after fornix injury. To gain more insight into the lesion-induced expression of GIP and its receptor (GIPR), expression profiles of the mRNAs were studied after rat sciatic nerve crush injury in 1) affected lumbar dorsal root ganglia (DRG), 2) spinal cord segments, and 3) proximal and distal nerve fragments by means of quantitative RT-PCR. Our results clearly identified lesion-induced as well as tissue type-specific mRNA regulation of GIP and its receptor. Furthermore, comprehensive immunohistochemical stainings not only confirmed and exceeded the previous observation of neuronal GIP expression but also revealed corresponding GIPR expression, implying putative modulatory functions of GIP/GIPR signaling in adult neurons. In complement, we also observed expression of GIP and its receptor in myelinating Schwann cells and oligodendrocytes. Polarized localization of GIPR in the abaxonal Schwann cell membranes, plasma membrane-associated GIPR expression of satellite cells, and ependymal GIPR expression strongly suggests complex cell type-specific functions of GIP and GIPR in the adult nervous system that are presumably mediated by autocrine and paracrine interactions, respectively. Notably, in vivo analyses with GIPR-deficient mice suggest a critical role of GIP/GIPR signal transduction in promoting spontaneous recovery after nerve crush, insofar as traumatic injury of GIPR-deficient mouse sciatic nerve revealed impaired axonal regeneration compared with wild-type mice.

Rôles des protéines Staufen 1 et 2 dans la plasticité synaptique des cellules pyramidales hippocampiques

La mémoire et l’apprentissage sont des phénomènes complexes qui demeurent encore incertains quant aux origines cellulaire et moléculaire. Il est maintenant connu que des changements au niveau des synapses, comme la plasticité synaptique, pourraient déterminer la base cellulaire de la formation de la mémoire. Alors que la potentialisation à long-terme (LTP) représente un renforcement de l’efficacité de transmission synaptique, la dépression à long-terme (LTD) constitue une diminution de l’efficacité des connexions synaptiques. Des études ont mis à jour certains mécanismes qui participent à ce phénomène de plasticité synaptique, notamment, les mécanismes d’induction et d’expression, ainsi que les changements morphologiques des épines dendritiques. La grande majorité des synapses excitatrices glutamatergiques se situe au niveau des épines dendritiques et la présence de la machinerie traductionnelle près de ces protubérances suggère fortement l’existence d’une traduction locale d’ARNm. Ces ARNm seraient d’ailleurs acheminés dans les dendrites par des protéines pouvant lier les ARNm et assurer leur transport jusqu’aux synapses activées. Le rôle des protéines Staufen (Stau1 et Stau2) dans le transport, la localisation et dans la régulation de la traduction de certains ARNm est bien établi. Toutefois, leur rôle précis dans la plasticité synaptique demeure encore inconnu. Ainsi, cette thèse de doctorat évalue l’importance des protéines Staufen pour le transport et la régulation d’ARNm dans la plasticité synaptique. Nous avons identifié des fonctions spécifiques à chaque isoforme; Stau1 et Stau2 étant respectivement impliquées dans la late-LTP et la LTD dépendante des récepteurs mGluR. Cette spécificité s’applique également au rôle que chaque isoforme joue dans la morphogenèse des épines dendritiques, puisque Stau1 semble nécessaire au maintien des épines dendritiques matures, alors que Stau2 serait davantage impliquée dans le développement des épines. D’autre part, nos travaux ont permis de déterminer que la morphogenèse des épines dendritiques dépendante de Stau1 était régulée par une plasticité synaptique endogène dépendante des récepteurs NMDA. Finalement, nous avons précisé les mécanismes de régulation de l’ARNm de la Map1b par Stau2 et démontré l’importance de Stau2 pour la production et l’assemblage des granules contenant les transcrits de la Map1b nécessaires pour la LTD dépendante des mGluR. Les travaux de cette thèse démontrent les rôles spécifiques des protéines Stau1 et Stau2 dans la régulation de la plasticité synaptique par les protéines Stau1 et Stau2. Nos travaux ont permis d’approfondir les connaissances actuelles sur les mécanismes de régulation des ARNm par les protéines Staufen dans la plasticité synaptique. MOTS-CLÉS EN FRANÇAIS: Staufen, hippocampe, plasticité synaptique, granules d’ARN, traduction, épines dendritiques.

Molecular characterization of Arabidopsis thaliana PUF proteins - binding specificity and target candidates

PUF proteins regulate both stability and translation through sequence-specific binding to the 3` UTR of target mRNA transcripts. Binding is mediated by a conserved PUF domain, which contains eight repeats of approximately 36 amino acids each. Found in all eukaryotes, they have been related to several developmental processes. Analysis of the 25 Arabidopsis Pumilio (APUM) proteins presenting PUF repeats reveals that 12 (APUM-1 to APUM-12) have a PUF domain with 50-75% similarity to the Drosophila PUF domain. Through three-hybrid assays, we show that APUM-1 to APUM-6 can bind specifically to the Nanos response element sequence recognized by Drosophila Pumilio. Using an Arabidopsis RNA library in a three-hybrid screening, we were able to identify an APUM-binding consensus sequence. Computational analysis allowed us to identify the APUM-binding element within the 3` UTR in many Arabidopsis transcripts, even in important mRNAs related to shoot stem cell maintenance. We demonstrate that APUM-1 to APUM-6 are able to bind specifically to APUM-binding elements in the 3` UTR of WUSCHEL, CLAVATA-1, PINHEAD/ZWILLE and FASCIATA-2 transcripts. The results obtained in the present study indicate that the APUM proteins may act as regulators in Arabidopsis through an evolutionarily conserved mechanism, which may open up a new approach for investigating mRNA regulation in plants.

Expressão dos fatores de crescimento fibroblástico 17 e 18 (FGF-17 e FGF-18) em folículos antrais bovinos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Polymorphic Sites at the 3 ' Untranslated Region of the HLA-G Gene Are Associated with Differential hla-g Soluble Levels in the Brazilian and French Population

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

DNA damage enhances melanogenesis.

Although the ability of UV irradiation to induce pigmentation in vivo and in vitro is well documented, the intracellular signals that trigger this response are poorly understood. We have recently shown that increasing DNA repair after irradiation enhances UV-induced melanization. Moreover, addition of small DNA fragments, particularly thymine dinucleotides (pTpT), selected to mimic sequences excised during the repair of UV-induced DNA photoproducts, to unirradiated pigment cells in vitro or to guinea pig skin in vivo induces a pigment response indistinguishable from UV-induced tanning. Here we present further evidence that DNA damage and/or the repair of this damage increases melanization. (i) Treatment with the restriction enzyme Pvu II or the DNA-damaging chemical agents methyl methanesulfonate (MMS) or 4-nitroquinoline 1-oxide (4-NQO) produces a 4- to 10-fold increase in melanin content in Cloudman S91 murine melanoma cells and an up to 70% increase in normal human melanocytes, (ii) UV irradiation, MMS, and pTpT all upregulate the mRNA level for tyrosinase, the rate-limiting enzyme in melanin biosynthesis. (iii) Treatment with pTpT or MMS increases the response of S91 cells to melanocyte-stimulating hormone (MSH) and increases the binding of MSH to its cell surface receptor, as has been reported for UV irradiation. Together, these data suggest that UV-induced DNA damage and/or the repair of this damage is an important signal in the pigmentation response to UV irradiation. Because Pvu II acts exclusively on DNA and because MMS and 4-NQO, at the concentrations used, primarily interact with DNA, such a stimulus alone appears sufficient to induce melanogenesis. Of possible practical importance, the dinucleotide pTpT mimics most, if not all, of the effects of UV irradiation on pigmentation, tyrosinase mRNA regulation, and response to MSH without the requirement for antecedent DNA damage.

Identificação e caracterização de RNA mensageiros candidatos a alvo das proteínas PUMILHO de Arabidopsis através do sistema triplo-híbrido de levedura

Proteínas PUF regulam a estabilidade e a tradução através da ligação a seqüências específicas nas regiões 3\' não traduzidas (3\' UTR) dos mensageiros. A ligação é mediada por um domínio de ligação conservado constituído por 8 repetições de aproximadamente 36 aminoácidos cada. Experimentos realizados no sistema triplo-híbrido de levedura mostraram que os homólogos PUF de Arabidopsis APUM-1, APUM-2 e APUM-3 são capazes de ligar especificamente à seqüência chamada de Elemento de Resposta a NANOS (NRE) reconhecida pelo homólogo PUF de Drosophila. A utilização de bibliotecas de expressão de RNA em ensaios no sistema triplo-híbrido permitiu a identificação de seqüências de ligação consenso para as três proteínas APUM. Análises computacionais identificaram elementos de ligação a APUM em regiões 3\' UTR de importantes transcritos relacionados ao controle do meristema do caule e à manutenção das células totipotentes. Nós mostramos que os homólogos APUM-l, APUM-2 e APUM-3 reconhecem elementos de ligação a APUM nas regiões 3\' UTR dos transcritos WUSCHEL, CLAVATA-1, ZWILLE e FASCIATA-2. Ensaios de RT-PCR e Western blot semiquantitativos mostraram que a quantidade dos transcritos WUSHEL e CLAVATA-1 é alterada em plantas antisenso induzíveis para APUM-l, APUM-2 e APUM-3. A relevância biológica dessas interações foi observada através de ensaios de coimunoprecipitação, confirmando, portanto, o primeiro caso de regulação traducional descrito para os mensageiros WUSCHEL e CLAVATA-1. Análises computacionais adicionais para a identificação de outros homólogos PUF em Arabidopsis encontraram vinte e cinco proteínas possuindo repetições PUF. Entre elas, os homólogos APUM-4, APUM-S e APUM-6 apresentam alta similaridade com as proteínas APUM-l, APUM-2 e APUM-3, sendo capazes de ligar especificamente à seqüência NRE e aos elementos de ligação a APUM presentes nas regiões 3\' UTR dos transcritos WUSCHEL, CLAVATA-1, ZWILLE e FASCIATA-ts resultados indicam que vários homólogos PUF podem agir como reguladores traducionais em Arabidopsis através de um mecanismo molecular conservado entre as espécies, podendo abrir uma nova área de investigação da regulação de mRNA em plantas.

Cardiac expression of the cystic fibrosis transmembrane conductance regulator involves novel Exon 1 usage to produce a unique amino-terminal protein

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel present in many cells. In cardiomyocytes, we report that multiple exon 1 usage and alternative splicing produces four CFTR transcripts, with different 5'-untranslated regions, CFTRTRAD-139, CFTR-1C/-1A, CFTR-1C, and CFTR-1B. CFTR transcripts containing the novel upstream exons (exons -1C, -1B, and -1A) represent more than 90% of cardiac expressed CFTR mRNA. Regulation of cardiac CFTR expression, in response to developmental and pathological stimuli, is exclusively due to the modulation of CFTR-1C and CFTR-1C/-1A expression. Upstream open reading frames have been identified in the 5'-untranslated regions of all CFTR transcripts that, in conjunction with adjacent stem-loop structures, modulate the efficiency of translation initiation at the AUG codon of the main CFTR coding region in CFTRTRAD-139 and CFTR-1C/-1A transcripts. Exon(-1A), only present in CFTR-1C/-1A transcripts, encodes an AUG codon that is in-frame with the main CFTR open reading frame, the efficient translation of which produces a novel CFTR protein isoform with a curtailed amino terminus. As the expression of this CFTR transcript parallels the spatial and temporal distribution of the cAMP-activated whole-cell current density in normal and diseased hearts, we suggest that CFTR-1C/-1A provides the molecular basis for the cardiac cAMP-activated chloride channel. Our findings provide further insight into the complex nature of in vivo CFTR expression, to which multiple mRNA transcripts, protein isoforms, and post-transcriptional regulatory mechanisms are now added.

Identification de protéines impliquées dans la localisation des ARNm au niveau de l'appareil mitotique

La localisation des ARNm au niveau des microtubules et des centrosomes laisse voir le centrosome et le fuseau mitotique comme des complexes ribonucléoprotéiques. Cependant, le mécanisme de localisation des ARNm à ces différentes structures ainsi que leurs fonctions dans la régulation de la mitose restent encore incompris. L’objectif était ici de caractériser des protéines de liaison à l’ARN (RNA Binding Proteins, RBPs) fonctionnellement impliquées dans la localisation des ARNm mitotiques chez la Drosophile et d’évaluer la conservation de la fonction de ces RBPs dans les cellules humaines. La déplétion de RBPs par RNAi générée dans des Drosophiles mutantes résulte en des phénotypes distincts de localisation anormale de l’ARNm centrosomique cen et en des défauts mitotiques différents selon le RBP ciblé, suggérant des fonctions différentes de ces RBPs. De plus, dans les jeunes embryons, les RBPs Bru-2 et Mask semblent être fonctionnellement importants pour la mitose via la régulation de l’ARNm cen, donnant un aperçu de la possible fonction mitotique de RBPs dans la régulation d’un ARN centrosomique. De plus, il a été observé dans un criblage d’immunofluorescence dans des cellules HeLa en métaphase que HNRNPUL1 colocalise au fuseau et aux centrosomes. HNRNPUL1 pourrait être impliqué dans la régulation de l’ARNm CDR2 (orthologue de cen) puisque la déplétion de l’orthologue de HNRNPUL1 dans la Drosophile, CG30122, résulte en une localisation anormale de l’ARNm centrosomique cen.

Identification de protéines impliquées dans la localisation des ARNm au niveau de l'appareil mitotique

La localisation des ARNm au niveau des microtubules et des centrosomes laisse voir le centrosome et le fuseau mitotique comme des complexes ribonucléoprotéiques. Cependant, le mécanisme de localisation des ARNm à ces différentes structures ainsi que leurs fonctions dans la régulation de la mitose restent encore incompris. L’objectif était ici de caractériser des protéines de liaison à l’ARN (RNA Binding Proteins, RBPs) fonctionnellement impliquées dans la localisation des ARNm mitotiques chez la Drosophile et d’évaluer la conservation de la fonction de ces RBPs dans les cellules humaines. La déplétion de RBPs par RNAi générée dans des Drosophiles mutantes résulte en des phénotypes distincts de localisation anormale de l’ARNm centrosomique cen et en des défauts mitotiques différents selon le RBP ciblé, suggérant des fonctions différentes de ces RBPs. De plus, dans les jeunes embryons, les RBPs Bru-2 et Mask semblent être fonctionnellement importants pour la mitose via la régulation de l’ARNm cen, donnant un aperçu de la possible fonction mitotique de RBPs dans la régulation d’un ARN centrosomique. De plus, il a été observé dans un criblage d’immunofluorescence dans des cellules HeLa en métaphase que HNRNPUL1 colocalise au fuseau et aux centrosomes. HNRNPUL1 pourrait être impliqué dans la régulation de l’ARNm CDR2 (orthologue de cen) puisque la déplétion de l’orthologue de HNRNPUL1 dans la Drosophile, CG30122, résulte en une localisation anormale de l’ARNm centrosomique cen.

Expression of substance P and preprotachykinin mRNA by primary sensory neurons in culture: regulation by factors present in peripheral and central target tissues.

Primary sensory neurons display various neuronal phenotypes which may be influenced by factors present in central or peripheral targets. In the case of DRG cells expressing substance P (SP), the influence of peripheral or central targets was tested on the neuronal expression of this neuropeptide. DRG cells were cultured from chick embryo at E6 or E10 (before or after establishment of functional connections with targets). Preprotachykinin mRNA was visualized in DRG cell cultures by either Northern blot or in situ hybridization using an antisense labeled riboprobe, while the neuropeptide SP was detected by immunostaining with a monoclonal antibody. In DRG cell cultures from E10, only 60% of neurons expressed SP. In contrast, DRG cell cultures performed at E6 showed a significant hybridization signal and SP-like immunoreactivity in virtually all the neurons (98%). The addition of extracts from muscle, skin, brain or spinal cord to DRG cells cultured at E6 reduced by 20% the percentage of neurons which express preprotachykinin mRNA and SP-like immunoreactivity. Our results indicate that factors issued from targets inhibit SP-expression by a subset of primary sensory neurons and act on the transcriptional control of preprotachykinin gene.