Mutations in the Drosophila rough deal gene affecting RZZ kinetochore function

International audienceBackground. The RZZ complex, composed of the proteins Rough-Deal (Rod), Zw10 and Zwilch, plays a central role in the spindle assembly checkpoint (SAC), which assures proper sister chromatid segregation during mitosis. RZZ contributes to the regulation of the spindle assembly checkpoint by helping to recruit Mad1-Mad2 and the microtubule motor dynein to unattached kinetochores. It is an important component of the outer kinetochore and specifically the fibrous corona whose expansion is believed to facilitate microtubule capture. How RZZ carries out its diverse activities is only poorly understood. The C-terminal region of the Rod subunit is relatively well-conserved across metazoan phylogeny, but no function has been attributed to it. Results. To explore the importance of the Rod_C domain in RZZ function in Drosophila, we generated a series of point mutations in a stretch of 200 residues within this domain and we report here their phenotypes. Several of the mutations profoundly disrupt recruitment of RZZ to kinetochores, including one in a temperature-sensitive manner, while still retaining the capacity to assemble into a complex with Zw10 and Zwilch. Others affect aspects of dynein activity or recruitment at the kinetochore. Conclusions and Significance. These results suggest that the Rod_C domain participates in the protein interactions necessary for RZZ recruitment and functionality at kinetochores

Inducing "cytokinesis" without mitosis in unfertilized Drosophila eggs.

International audienceSelection of the cleavage plane during cytokinesis in dividing cells is linked to the position of the mitotic spindle. A major player in cleavage plane positioning is believed to be the anaphase central spindle and its associated signaling complex called centralspindlin, composed of MgcRacGap and MKLP1. Centralspindlin has the capacity to induce furrowing of the cell cortex by promoting the localized activation of RhoA, which in turn promotes assembly of the contractile ring. We have found a way to induce a cytokinesis-like process in unfertilized Drosophila eggs and very early embryos, when spindle structures are few and located far from invaginating egg cortex. The simple injection of a small molecule inhibitor of Cdk1/Cyclin B (either Roscovitin or RO3306) is sufficient to promote membrane invagination near the site of injection. The furrow generated is in many respects similar to a classical cleavage furrow. Actin, myosin, anillin and MKLP1 are all associated with the forming furrow, which in some cases can entirely circumscribe the unfertilized egg. A similar furrow can also be generated by the localized injection of constitutively active RhoA protein, suggesting that Cdk1 is normally an upstream inhibitor of RhoA activation. We show further that this process apparently is not associated with microtubules. Since simple localized inhibition of Cdk1 is sufficient to induce a furrow, we suggest that in real cytokinesis in normal cells, the localized downregulation of Cdk1 activity at the metaphase-anaphase transition may contribute, along with the spindle, to the positioning of the cleavage furrow

Determinants of the ubiquitin-mediated degradation of the Met4 transcription factor.

In yeast, the Met4 transcription factor and its cofactors Cbf1, Met28, Met31, and Met32 control the expression of sulfur metabolism and oxidative stress response genes. Met4 activity is tuned to nutrient and oxidative stress conditions by the SCF(Met30) ubiquitin ligase. The mechanism whereby SCF(Met30)-dependent ubiquitylation of Met4 controls Met4 activity remains contentious. Here, we have demonstrated that intracellular cysteine levels dictate the degradation of Met4 in vivo, as shown by the ability of cysteine, but not methionine or S-adenosylmethionine (AdoMet), to trigger Met4 degradation in an str4Delta strain, which lacks the ability to produce cysteine from methionine or AdoMet. Met4 degradation requires its nuclear localization and activity of the 26 S proteasome. Analysis of the regulated degradation of a fully functional Met4-Cbf1 chimera, in which Met4 is fused to the DNA binding domain of Cbf1, demonstrates that elimination of Met4 in vivo can be triggered independently of both its normal protein interactions. Strains that harbor the Met4-Cbf1 fusion as the only source of Cbf1 activity needed for proper kinetochore function exhibit high rates of methionine-dependent chromosomal instability. We suggest that SCF(Met30) activity or Met4 utilization as a substrate may be directly regulated by intracellular cysteine concentrations

A dual, catalytic role for the fission yeast Ccr4-Not complex in gene silencing and heterochromatin spreading

Heterochromatic gene silencing relies on combinatorial control by specific histone modifications, the occurrence of transcription, and/or RNA degradation. Once nucleated, heterochromatin propagates within defined chromosomal regions and is maintained throughout cell divisions to warrant proper genome expression and integrity. In the fission yeast Schizosaccharomyces pombe, the Ccr4-Not complex partakes in gene silencing, but its relative contribution to distinct heterochromatin domains and its role in nucleation versus spreading have remained elusive. Here, we unveil major functions for Ccr4-Not in silencing and heterochromatin spreading at the mating type locus and subtelomeres. Mutations of the catalytic subunits Caf1 or Mot2, involved in RNA deadenylation and protein ubiquitinylation, respectively, result in impaired propagation of H3K9me3 and massive accumulation of nucleation-distal heterochromatic transcripts. Both silencing and spreading defects are suppressed upon disruption of the heterochromatin antagonizing factor Epe1. Overall, our results position the Ccr4-Not complex as a critical, dual regulator of heterochromatic gene silencing and spreading

Ubiquitylation of the COMPASS component Swd2 links H2B ubiquitylation to H3K4 trimethylation.

International audienceMono-ubiquitylation of histone H2B correlates with transcriptional activation and is required for di- and trimethylation at Lys 4 on the histone H3 tail (H3K4) by the SET1/COMPASS methyltransferase complex through a poorly characterized trans-tail pathway. Here we show that mono-ubiquitylation of histone H2B promotes ubiquitylation at Lys 68 and Lys 69 of Swd2, the essential component of SET1/COMPASS in Saccharomyces cerevisiae. We found that Rad6/Bre1 ubiquitylation enzymes responsible for H2B ubiquitylation also participate directly in Swd2 modification. Preventing Swd2 or H2B ubiquitylation did not affect Set1 stability, interaction of Swd2 with Set1 or the ability of Swd2 to interact with chromatin. However, we found that mutation of Lys 68 and Lys 69 of Swd2 markedly reduced trimethylation, and to a lesser extent dimethylation, of H3K4 at the 5'-end of transcribing genes without affecting monomethylation. This effect results from the ability of Swd2 ubiquitylation to control recruitment of Spp1, a COMPASS subunit necessary for trimethylation. Our results further indicate that Swd2 is a major H3-binding component of COMPASS. Swd2 thus represents a key factor that mediates crosstalk between H2B ubiquitylation and H3K4 trimethylation on chromatin

Régulation des émotions chez l'animal d'élevage : focus sur les acteurs neurobiologiques

Dossier : Neurobiologie des fonctions et des comportementsThe interest for emotion in farm animals is motivated by the aim to improve welfare in livestock production. The study of emotions is based on the cognitive capacities of animals to perceive, evaluate and react to their environment. The perception capacity involves different senses, allows the animal to recognize its environment, and not necessarily induces emotion. Emotions are induced by specific situations as novelty, social separation or predator. Moreover, emotional reactions, evaluated by behavioural, endocrine and neurovegetative responses, are modulated by gender, physiological state or some hormones such as melatonin. The brain structures of the neuronal network of emotions are involved in perception, integrative levels and expression of emotional responses. In farm animals, the most studied brain structures are those of the olfactory system for perception, the frontal cortex and amygdala for integration of the environment, the paraventricular nucleus of the hypothalamus for endocrine responses. Other studies hypothesize the role of the brainstem nuclei in neurovegetative responses, and those of the periaqueductal grey for its putative role in behavioural responses and more generally in the coping strategy. In perspective, it is necessary to better understand the development of the neuronal network of emotions in farm animals, especially the effect of early experience, and the cognitive evaluation involved in emotion.La question des émotions des animaux d’élevage s’inscrit dans la volonté d’améliorer les conditions d’élevage en prenant en compte leur bien-être. La genèse des émotions repose sur la capacité cognitive des individus à percevoir, évaluer et réagir à leur environnement. La capacité de perception met en jeu différentes sensorialités et participe à la reconnaissance des éléments de l’environnement de l’animal sans nécessairement déclencher d’émotions. Les situations déclenchantes d’émotion les plus étudiées chez les animaux d’élevage sont la nouveauté, la séparation sociale ou la présence d’un être humain. De plus, les réactions émotionnelles, évaluées par les réponses comportementales, endocriniennes et neurovégétatives sont modulées par le genre, l’état physiologique ou certaines hormones comme la mélatonine. Les structures cérébrales du réseau neuronal des émotions sont impliquées dans la perception et le traitement des informations de l’environnement, et/ou dans l’expression des réponses émotionnelles. Chez les animaux d’élevage, les structures cérébrales les plus étudiées sont le système olfactif pour la perception, le cortex frontal et l’amygdale pour le traitement des informations, le noyau paraventriculaire de l’hypothalamus pour les réponses endocriniennes. D’autres travaux suggèrent le rôle potentiel de structures du tronc cérébral dans les réponses neurovégétatives et de la substance grise périaqueducale dans les réponses comportementales et plus largement dans les stratégies d’adaptation. En perspective, il est maintenant nécessaire d’étudier l’impact de l’expérience précoce sur le développement du réseau neuronal des émotions et de mieux comprendre la part de l’évaluation cognitive dans la genèse des émotions

A scaffold lncRNA shapes the mitosis to meiosis switch

International audienceLong non-coding RNAs (lncRNAs) contribute to the regulation of gene expression in response to intra-or extracellular signals but the underlying molecular mechanisms remain largely unexplored. Here, we identify an uncharacterized lncRNA as a central player in shaping the meiotic gene expression program in fission yeast. We report that this regulatory RNA, termed mamRNA, scaffolds the antagonistic RNA-binding proteins Mmi1 and Mei2 to ensure their reciprocal inhibition and fine tune meiotic mRNA degradation during mitotic growth. Mechanistically, mamRNA allows Mmi1 to target Mei2 for ubiquitin-mediated downregulation, and conversely enables accumulating Mei2 to impede Mmi1 activity, thereby reinforcing the mitosis to meiosis switch. These regulations also occur within a unique Mmi1containing nuclear body, positioning mamRNA as a spatially-confined sensor of Mei2 levels. Our results thus provide a mechanistic basis for the mutual control of gametogenesis effectors and further expand our vision of the regulatory potential of lncRNAs