The genomic repertoire for cell cycle control and DNA metabolism in S. purpuratus

A search of the Strongylocentrotus purpuratus genome for genes associated with cell cycle control and DNA metabolism shows that the known repertoire of these genes is conserved in the sea urchin, although with fewer family members represented than in vertebrates, and with some cases of echinoderm-specific gene diversifications. For example, while homologues of the known cyclins are mostly encoded by single genes in S. purpuratus (unlike vertebrates, which have multiple isoforms), there are additional genes encoding novel cyclins of the B and K/L types. Almost all known cyclin-dependent kinases (CDKs) or CDK-like proteins have an orthologue in S. purpuratus; CDK3 is one exception, whereas CDK4 and 6 are represented by a single homologue, referred to as CDK4. While the complexity of the two families of mitotic kinases, Polo and Aurora, is close to that found in the nematode, the diversity of the NIMA-related kinases (NEK proteins) approaches that of vertebrates. Among the nine NEK proteins found in S. purpuratus, eight could be assigned orthologues in vertebrates, whereas the ninth is unique to sea urchins. Most known DNA replication, DNA repair and mitotic checkpoint genes are also present, as are homologues of the pRB (two) and p53 (one) tumor suppressors. Interestingly, the p21/p27 family of CDK inhibitors is represented by one homologue, whereas the INK4 and ARF families of tumor suppressors appear to be absent, suggesting that these evolved only in vertebrates. Our results suggest that, while the cell cycle control mechanisms known from other animals are generally conserved in sea urchin, parts of the machinery have diversified within the echinoderm lineage. The set of genes uncovered in this analysis of the S. purpuratus genome should enhance future research on cell cycle control and developmental regulation in this model

Marine Natural Meroterpenes: Synthesis and Antiproliferative Activity

Meroterpenes are compounds of mixed biogenesis, isolated from plants, microorganisms and marine invertebrates. We have previously isolated and determined the structure for a series of meroterpenes extracted from the ascidian Aplidium aff. densum. Here, we demonstrate the chemical synthesis of three of them and their derivatives, and evaluate their biological activity on two bacterial strains, on sea urchin eggs, and on cancerous and healthy human cells

Structures and activities of tiahuramides A–C, cyclic depsipeptides from a tahitian collection of the marine cyanobacterium Lyngbya majuscula

The structures of three new cyclic depsipeptides, tiahuramides A (1), B (2), and C (3), from a French Polynesian collection of the marine cyanobacterium Lyngbya majuscula are described. The planar structures of these compounds were established by a combination of mass spectrometry and 1D and 2D NMR experiments. Absolute configurations of natural and nonproteinogenic amino acids were determined through a combination of acid hydrolysis, derivitization with Marfey’s reagent, and HPLC. The absolute configuration of hydroxy acids was confirmed by Mosher’s method. The antibacterial activities of tiahuramides against three marine bacteria were evaluated. Compound 3 was the most active compound of the series, with an MIC of 6.7 μM on one of the three tested bacteria. The three peptides inhibit the first cell division of sea urchin fertilized eggs with IC50 values in the range from 3.9 to 11 μM. Tiahuramide B (2), the most potent compound, causes cellular alteration characteristics of apoptotic cells, blebbing, DNA condensation, and fragmentation, already at the first egg cleavage. The cytotoxic activity of compounds 1–3 was tested in SH-SY5Y human neuroblastoma cells. Compounds 2 and 3 showed an IC50 of 14 and 6.0 μM, respectively, whereas compound 1 displayed no toxicity in this cell line at 100 μM. To determine the type of cell death induced by tiahuramide C (3), SH-SY5Y cells were costained with annexin V–FITC and propidium iodide and analyzed by flow cytometry. The double staining indicated that the cytotoxicity of compound 3 in this cell line is produced by necrosis

Embryon d'oursin et séquençage du génome de l'espèce

Depuis longtemps, l'oursin est un modèle privilégié pour les recherches sur le développement. De plus, la fécondation et la croissance externe des embryons, leur cycle de division rapide ainsi que leur transparence, appropriée aux techniques de visualisation moléculaire les plus actuelles, font également de l'embryogenèse précoce de l'oursin un modèle de choix pour l'analyse des mécanismes qui régulent la division cellulaire. Ces caractéristiques, ainsi que la position phylogénétique de l'oursin, proche des vertébrés et cependant dans un groupe externe, ont conduit la communauté scientifique travaillant sur ce modèle à séquencer très récemment le génome complet de l'espèce Strongylo-centrotus purpuratus. Ce génome contient un répertoire pratiquement complet des gènes régulateurs de la division cellulaire. La comparaison avec les répertoires équivalents de vertébrés, d'insectes, de nématodes ou de tuniciers donne un nouvel éclairage à l'évolution du contrôle du cycle cellulaire. Chaque famille de gènes comporte chez l'oursin un nombre limité de composants. Chez les vertébrés, de nombreuses familles ont subi une forte expansion (cyclines, kinases mitotiques...), néanmoins d'autres gènes semblent être absents, comme par exemple une nouvelle cycline de type B mise en évidence chez l'oursin. Par ailleurs, certains gènes, que l'on croyait jusque là spécifiques des vertébrés, existent également chez S. purpuratus (MCM9,...). Enfin, il est important de noter l'absence chez l'oursin des inhibiteurs du cycle cellulaire de la famille des INK, qui seraient donc vraisemblablement spécifiques des vertébrés. Cet apport considérable de nouveaux outils moléculaires pour l'oursin donnera sans aucun doute un nouvel essor aux travaux concernant les mécanismes de division dans cette espèce

CDK13, a Kinase Involved in Pre-mRNA Splicing, Is a Component of the Perinucleolar Compartment

<div><p>The perinucleolar compartment (PNC) is a subnuclear stucture forming predominantly in cancer cells; its prevalence positively correlates with metastatic capacity. Although several RNA-binding proteins have been characterized in PNC, the molecular function of this compartment remains unclear. Here we demonstrate that the cyclin–dependent kinase 13 (CDK13) is a newly identified constituent of PNC. CDK13 is a kinase involved in the regulation of gene expression and whose overexpression was found to alter pre-mRNA processing. In this study we show that CDK13 is enriched in PNC and co-localizes all along the cell cycle with the PNC component PTB. In contrast, neither the cyclins K and L, known to associate with CDK13, nor the potential kinase substrates accumulate in PNC. We further show that CDK13 overexpression increases PNC prevalence suggesting that CDK13 may be determinant for PNC formation. This result linked to the finding that CDK13 gene is amplified in different types of cancer indicate that this kinase can contribute to cancer development in human.</p></div

Localization of Cyclins K and L in interphase.

<p>GFP-cyclin K and HA-cyclins L1 and L2 were expressed in HeLa cells and their localizations were analysed by fluorescence microscopy using respectively GFP-cyclin K (A) or immunofluorescence with anti-cyclin L1 (D) and L2 (G) antibodies. Localizations were compared with the one of endogenous PTB (B,E,H). Respective merge images (C,F,I) show an absence or a very poor co-localization of cyclins with CDK13 in PNC. An increased magnification of merge labelling is inserted in C and I. Scale bar: 5μm.</p

Increased CDK13 complexes expression decreases PNC prevalence.

<p>(A) GFP-tagged cyclins were expressed with or without HA-CDK13 in HeLa cells and the level of protein expression was controlled by GFP fluorescence or immunofluorescence. PNC prevalence was measured by immunofluorescence labelling with anti-PTB antibodies. Percentages (± SD) of transfected (GFP-positive) and non-transfected (nt) cells containing nuclear PTB-positive dots were evaluated by counting for each condition 500 cultured cells in three different transfection experiments. In those experiments, 30% of non-transfected HeLa cells were PTB-positive. **P<0.001. (B) HeLa cells were treated with (or without) siRNAs targeting CDK13. SiRNA2 only faintly diminished CDK13 expression levels and did not significantly altered PNC prevalence. In contrast, siRNA5 strongly altered CDK13 expression, leading to a significant decrease in PNC prevalence. In this set of experiments, 50% of control cells (w/o siRNA) were PTB-positive. *P<0,05.</p

Clk2 is not present in PNC.

<p>Clk2, revealed with antibodies to the GST-tag (B,D) and GFP-CDK13 (A) localized in common foci in the nucleoplasm as shown in merge picture (C) while Clk2 is not present in PNC as visualized with PTB colabelling (D-F). Scale bar: 5μm.</p

CDC2L5, a Cdk-like kinase with RS domain, interacts with the ASF/SF2-associated protein p32 and affects splicing in vivo

International audienceThe human CDC2L5 gene encodes a protein of unknown physiological function. This protein is closely related to the cyclin-dependent kinase (Cdks) family and contains an arginine/serine-rich (RS) domain. The Cdks were first identified as crucial regulators of cell-cycle progression, more recently they were found to be involved in transcription and mRNA processing. RS domains are mainly present in proteins regulating pre-mRNA splicing, suggesting CDC2L5 having a possible role in this process. In this study, we demonstrate that CDC2L5 is located in the nucleoplasm, at a higher concentration in speckles, the storage sites for splicing factors. Furthermore, this localization is dependent on the presence of the N-terminal sequence including the RS domain. Then, we report that CDC2L5 directly interacts with the ASF/SF2-associated protein p32, a protein involved in splicing regulation. Overexpression of CDC2L5 constructs disturbs constitutive splicing and switches alternative splice site selection in vivo. These results argue in favor of a functional role of the CDC2L5 kinase in splicing regulation

Localization of CDK13, nucleolin and fibrillarin during interphase.

<p>In HeLa cells, the localization of CDK13 (A,C,D,F), nucleolin (B,C) and fibrillarin (E,F) were detected using rabbit anti-CDK13 and mouse anti-nucleolin or anti-fibrillarin antibodies. A poor (C) or absent (F) colocalization of CDK13 with these nucleolar markers demonstrates that CDK13 is located in a perinucleolar structure. Scale bar: 5μm.</p