Cushing's Syndrome and Fetal Features Resurgence in Adrenal Cortex–Specific Prkar1a Knockout Mice

Carney complex (CNC) is an inherited neoplasia syndrome with endocrine overactivity. Its most frequent endocrine manifestation is primary pigmented nodular adrenocortical disease (PPNAD), a bilateral adrenocortical hyperplasia causing pituitary-independent Cushing's syndrome. Inactivating mutations in PRKAR1A, a gene encoding the type 1 α-regulatory subunit (R1α) of the cAMP–dependent protein kinase (PKA) have been found in 80% of CNC patients with Cushing's syndrome. To demonstrate the implication of R1α loss in the initiation and development of PPNAD, we generated mice lacking Prkar1a specifically in the adrenal cortex (AdKO). AdKO mice develop pituitary-independent Cushing's syndrome with increased PKA activity. This leads to autonomous steroidogenic genes expression and deregulated adreno-cortical cells differentiation, increased proliferation and resistance to apoptosis. Unexpectedly, R1α loss results in improper maintenance and centrifugal expansion of cortisol-producing fetal adrenocortical cells with concomitant regression of adult cortex. Our data provide the first in vivo evidence that loss of R1α is sufficient to induce autonomous adrenal hyper-activity and bilateral hyperplasia, both observed in human PPNAD. Furthermore, this model demonstrates that deregulated PKA activity favors the emergence of a new cell population potentially arising from the fetal adrenal, giving new insight into the mechanisms leading to PPNAD

ETUDE DES MECANISMES MOLECULAIRES IMPLIQUES DANS LE CONTROLE HORMONAL ET TISSULAIRE DE L'EXPRESSION DU GENE D'UNE ALDO-CETO REDUCTASE, MVDP/AKR1B7, DANS LE CORTEX SURRENALIEN ET DES LIGNEES CELLULAIRES STEROIDOGENES

LE GENE MVDP (MOUSE VAS DEFERENS PROTEIN) CODE POUR UNE ALDO-CETO REDUCTASE. IL EST EXPRIME DANS LE CORTEX SURRENALIEN DE DIFFERENTS RONGEURS OU SON EXPRESSION EST RESTREINTE A LA ZONE FASCICULEE. LA PROTEINE MVDP EST RESPONSABLE DE LA DETOXICATION DE L'ISOCAPROALDEHYDE, PRODUIT LORS DE LA 1 O ETAPE DE LA STEROIDOGENESE. SON EXPRESSION EST CONTROLEE PAR L'ACTH ESSENTIELLEMENT A UN NIVEAU TRANSCRIPTIONNEL, A L'IMAGE DES GENES DE LA STEROIDOGENESE. MVDP EST EGALEMENT RETROUVEE DANS D'AUTRES TISSUS STEROIDOGENES : OVAIRES ET TESTICULES, A DES NIVEAUX D'ACCUMULATION PLUS FAIBLES. LE CONTROLE HORMONAL DU GENE DANS LES CELLULES SURRENALIENNES ET TESTICULAIRES UTILISENT CEPENDANT DES MECANISMES DIFFERENTS. LES MECANISMES MOLECULAIRES DE CE CONTROLE ONT ETE ANALYSES PAR DES EXPERIENCES DE TRANSFECTIONS TRANSITOIRES ET DE RETARD SUR GEL. DANS DES CELLULES CORTICO-SURRENALIENNES Y1, LA PLUS PETITE REGION PROMOTRICE CAPABLE DE PRODUIRE UNE ACTIVITE BASALE ET INDUITE PAR L'AMPC EST LA REGION -121/+41. UN SITE DE LIAISON ATYPIQUE POUR LE FACTEUR SPECIFIQUE DES TISSUS STEROIDIENS SF-1 (102), ET UN SITE POUR NF1 (76), SONT NECESSAIRES A UNE ACTIVITE MAXIMALE DU PROMOTEUR. LA REPONSE HORMONALE REQUIERT UNE COOPERATION FONCTIONNELLE ENTRE LES SITES 61 ET 52 FIXANT, RESPECTIVEMENT, LES FACTEURS C/EBP ET SP1. FINALEMENT, DES ELEMENTS RESPONSABLES DE LA DIFFERENCE D'EXPRESSION DE MVDP DANS LES CELLULES DE LEYDIG ET CELLULES CORTICO-SURRENALIENNES SONT CONTENUS DANS LA REGION 510/+41. CETTE REGION FIXE DES COMPLEXES PROTEIQUES COMMUNS AUX CELLULES Y1 ET MA-10 CONTENANT LE FACTEUR SF-1 SOUS FORME DE MONOMERE (458) OU D'OLIGOMERE (503), AINSI QU'UN COMPLEXE NON IDENTIFIE SPECIFIQUE DES CELLULES SURRENALIENNES (503).CLERMONT FD-BCIU Sci.et Tech. (630142101) / SudocSudocFranceF

The translational regulator CPEB1 provides a link between dcp1 bodies and stress granules

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GW body disassembly triggered by siRNAs independently of their silencing activity

International audienceGW bodies (or P-bodies) are cytoplasmic granules containing proteins involved in both mRNA degradation and storage, including the RNA interference machinery. Their mechanism of assembly and function are still poorly known although their number depends upon the flux of mRNA to be stored or degraded. We show here that silencing of the translational regulator CPEB1 leads to their disappearance, as reported for other GW body components. Surprisingly, the same results were obtained with several siRNAs targeting genes encoding proteins unrelated to mRNA metabolism. The disappearance of GW bodies did not correlate with the silencing activity of the siRNA and did not inhibit further silencing by siRNA. Importantly, in most cases, GW bodies were rapidly reinduced by arsenite, indicating that their assembly was not prevented by the inhibition of the targeted or off-target genes. We therefore propose that some siRNA sequences affect mRNA metabolism so as to diminish the amount of mRNA directed to the GW bodies. As an exception, GW bodies were not reinduced following Rck/p54 depletion by interference, indicating that this component is truly required for the GW body assembly. Noteworthy, Rck/p54 was dispensable for the assembly of stress granules, in spite of their close relationship with the GW bodies

Steroidogenic Factor-1 Controls the Aldose Reductase akr1b7 Gene Promoter in Transgenic Mice through an Atypical Binding Site

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Adrenocorticotropin/3′,5′-Cyclic AMP-Mediated Transcription of the Scavenger akr1-b7 Gene in Adrenocortical Cells Is Dependent on Three Functionally Distinct Steroidogenic Factor-1-Responsive Elements

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Eukaryotic rRNA Modification by Yeast 5-Methylcytosine-Methyltransferases and Human Proliferation-Associated Antigen p120

International audienceModified nucleotide 5-methylcytosine (m(5)C) is frequently present in various eukaryotic RNAs, including tRNAs, rRNAs and in other non-coding RNAs, as well as in mRNAs. RNA: m(5)C-methyltranferases (MTases) Nop2 from S. cerevisiae and human proliferation-associated nucleolar antigen p120 are both members of a protein family called Nop2/NSUN/NOL1. Protein p120 is well-known as a tumor marker which is over-expressed in various cancer tissues. Using a combination of RNA bisulfite sequencing and HPLC-MS/MS analysis, we demonstrated here that p120 displays an RNA:m(5)C-MTase activity, which restores m(5)C formation at position 2870 in domain V of 25S rRNA in a nop2 Delta yeast strain. We also confirm that yeast proteins Nop2p and Rcm1p catalyze the formation of m(5)C in domains V and IV, respectively. In addition, we do not find any evidence of m(5)C residues in yeast 18S rRNA. We also performed functional complementation of Nop2-deficient yeasts by human p120 and studied the importance of different sequence and structural domains of Nop2 and p120 for yeast growth and m(5)C-MTase activity. Chimeric protein formed by Nop2 and p120 fragments revealed the importance of Nop2 N-terminal domain for correct protein localization and its cellular function. We also validated that the presence of Nop2, rather than the m(5)C modification in rRNA itself, is required for pre-rRNA processing. Our results corroborate that Nop2 belongs to the large family of pre-ribosomal proteins and possesses two related functions in pre-rRNA processing: as an essential factor for cleavages and m(5)C: RNA:modification. These results support the notion of quality control during ribosome synthesis by such modification enzymes

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images

International audienceThe last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Becaus

Enhanced SRSF5 Protein Expression Reinforces Lamin A mRNA Production in HeLa Cells and Fibroblasts of Progeria Patients

International audienceThe Hutchinson Gilford Progeria Syndrome (HGPS) is a rare genetic disease leading to accelerated aging. Three mutations of the LMNA gene leading to HGPS were identified. The more frequent ones, c.1824C>T and c.1822G>A, enhance the use of the intron 11 progerin 5splice site (5SS) instead of the LMNA 5SS, leading to the production of the truncated dominant negative progerin. The less frequent c.1868C>G mutation creates a novel 5SS (LA35 5SS), inducing the production of another truncated LMNA protein (LA35). Our data show that the progerin 5SS is used at low yield in the absence of HGPS mutation, whereas utilization of the LA35 5SS is dependent upon the presence of the c.1868C>G mutation. In the perspective to correct HGPS splicing defects, we investigated whether SR proteins can modify the relative yields of utilization of intron 11 5SSs. By in cellulo and in vitro assays, we identified SRSF5 as a direct key regulator increasing the utilization of the LMNA 5SS in the presence of the HGPS mutations. Enhanced SRSF5 expression in dermal fibroblasts of HGPS patients as well as PDGF-BB stimulation of these cells decreased the utilization of the progerin 5SS, and improves nuclear morphology, opening new therapeutic perspectives for premature aging. (C) 2015 Wiley Periodicals, Inc

Identification of protein partners of the human immunodeficiency virus 1 tat/rev exon 3 leads to the discovery of a new HIV-1 splicing regulator, protein hnRNP K.

HIV-1 pre-mRNA splicing depends upon 4 donor and 8 acceptor sites, which are used in combination to produce more than 40 different mRNAs. The acceptor site A7 plays an essential role for tat and rev mRNA production. The SLS2-A7 stem-loop structure containing site A7 was also proposed to modulate HIV-1 RNA export by the Rev protein. To further characterize nuclear factors involved in these processes, we purified RNP complexes formed by incubation of SLS2-A7 RNA transcripts in HeLa cell nuclear extracts by affinity chromatography and identified 33 associated proteins by nanoLC-MS/MS. By UV cross-linking, immunoselection and EMSA, we showed that, in addition to the well-known hnRNP A1 inhibitor of site A7, nucleolin, hnRNP H and hnRNP K interact directly with SLS2-A7 RNA. Nucleolin binds to a cluster of successive canonical NRE motifs in SLS2-A7 RNA, which is unique in HIV-1 RNA. Proteins hnRNP A1 and hnRNP K bind synergistically to SLS2-A7 RNA and both have a negative effect on site A7 activity. By the use of a plasmid expressing a truncated version of HIV-1 RNA, we showed a strong effect of the overexpression of hnRNP K in HeLa cells on HIV-1 alternative splicing. As a consequence, production of the Nef protein was strongly reduced. Interestingly also, many proteins identified in our proteomic analysis are known to modulate either the Rev activity or other mechanisms required for HIV-1 multiplication and several of them seem to be recruited by hnRNP K, suggesting that hnRNP K plays an important role for HIV-1 biology