DNA breaks are masked by multiple Rap1 binding in yeast: implications for telomere capping and telomerase regulation

Eukaryotic cells distinguish their chromosome ends from accidental DNA double-strand breaks by packaging them in a protective structure referred to as the telomere “cap.” Here we investigate the nature of the telomere cap by examining events at DNA breaks generated adjacent to either natural telomeric sequences (TG repeats) or arrays of Rap1-binding sites that vary in length. Although DNA breaks adjacent to either short or long telomeric sequences are efficiently converted into stable telomeres, they elicit very different initial responses. Short telomeric sequences (80 base pair [bp]) are avidly bound by Mre11, as well as the telomere capping protein Cdc13 and telomerase enzyme, consistent with their rapid telomerase-dependent elongation. Surprisingly, little or no Mre11 binding is detected at long telomere tracts (250 bp), and this is correlated with reduced Cdc13 and telomerase binding. Consistent with these observations, ends with long telomere tracts undergo strongly reduced exonucleolytic resection and display limited binding by both Rpa1 and Mec1, suggesting that they fail to elicit a checkpoint response. Rap1 binding is required for end concealment at long tracts, but Rif proteins, yKu, and Cdc13 are not. These results shed light on the nature of the telomere cap and mechanisms that regulate telomerase access at chromosome ends

Capping and de novo telomere addition at a DNA double-strand break: role of Tbf1p in Saccharomyces cerevisiae

Les télomères protègent les extrémités chromosomiques d'être reconnues comme des cassures double brin. Dans mon travail de thèse, j'ai étudié les mécanismes permettant de distinguer un télomère d'une cassure de l'ADN. Chez Saccharomyces cerevisiae, j'ai montré que de longues répétitions humaines de T2AG3 liées par Tbf1p sont cachées de la voie de détection des dommages de l'ADN. Une courte séquence est quant à elle efficacement allongée par la télomérase mais active transitoirement la voie de réparation de l'ADN. La capacité de Tbf1p de protéger les séquences télomériques T2AG3 est indépendante des deux protéines Vid22p et Ygr071cp interagissant avec Tbf1p. De manière surprenante, j'ai pu détecter la présence de la télomérase aux extrémités des cassures double brin de l'ADN malgré l'absence d'élongation. Enfin, j'ai démontré que Tbf1p, Vid22p et Ygr071cp sont recrutées à des cassures double brin suggérant qu'elles sont impliquées dans la réponse à un dommage de l'ADN

DNA-end capping by the budding yeast transcription factor and subtelomeric binding protein Tbf1

International audienc

Projection of the Gruneberg ganglion to the mouse olfactory bulb

In mammals, sensory neurons from the main olfactory and vomeronasal systems project their axons to the olfactory bulbs in the brain. We here report that a cluster of neurons, distinct from these two systems, located at the very tip of the mouse nose and called the Gruneberg ganglion expresses the mature olfactory-sensory neuron-specific marker olfactory marker protein (OMP), but is unlikely to express known odorant or pheromone receptors. The ganglion is present at birth and maintained during adult life. Tracing experiments indicate that these neurons target ipsilaterally to a specific set of glomeruli located on the caudal part of the olfactory bulb, and that this connection is necessary for the survival of the ganglion. The glomerular targets are structures previously proposed to be associated with suckling behaviour. These observations strongly suggest that this peculiar olfactory neuronal population plays a sensory role, possibly linked to chemoperception

The not5 subunit of the ccr4-not complex connects transcription and translation

Recent studies have suggested that a sub-complex of RNA polymerase II composed of Rpb4 and Rpb7 couples the nuclear and cytoplasmic stages of gene expression by associating with newly made mRNAs in the nucleus, and contributing to their translation and degradation in the cytoplasm. Here we show by yeast two hybrid and co-immunoprecipitation experiments, followed by ribosome fractionation and fluorescent microscopy, that a subunit of the Ccr4-Not complex, Not5, is essential in the nucleus for the cytoplasmic functions of Rpb4. Not5 interacts with Rpb4; it is required for the presence of Rpb4 in polysomes, for interaction of Rpb4 with the translation initiation factor eIF3 and for association of Rpb4 with mRNAs. We find that Rpb7 presence in the cytoplasm and polysomes is much less significant than that of Rpb4, and that it does not depend upon Not5. Hence Not5-dependence unlinks the cytoplasmic functions of Rpb4 and Rpb7. We additionally determine with RNA immunoprecipitation and native gel analysis that Not5 is needed in the cytoplasm for the co-translational assembly of RNA polymerase II. This stems from the importance of Not5 for the association of the R2TP Hsp90 co-chaperone with polysomes translating RPB1 mRNA to protect newly synthesized Rpb1 from aggregation. Hence taken together our results show that Not5 interconnects translation and transcription

The Not5 Subunit of the Ccr4-Not Complex Connects Transcription and Translation

Recent studies have suggested that a sub-complex of RNA polymerase II composed of Rpb4 and Rpb7 couples the nuclear and cytoplasmic stages of gene expression by associating with newly made mRNAs in the nucleus, and contributing to their translation and degradation in the cytoplasm. Here we show by yeast two hybrid and co-immunoprecipitation experiments, followed by ribosome fractionation and fluorescent microscopy, that a subunit of the Ccr4-Not complex, Not5, is essential in the nucleus for the cytoplasmic functions of Rpb4. Not5 interacts with Rpb4; it is required for the presence of Rpb4 in polysomes, for interaction of Rpb4 with the translation initiation factor eIF3 and for association of Rpb4 with mRNAs. We find that Rpb7 presence in the cytoplasm and polysomes is much less significant than that of Rpb4, and that it does not depend upon Not5. Hence Not5-dependence unlinks the cytoplasmic functions of Rpb4 and Rpb7. We additionally determine with RNA immunoprecipitation and native gel analysis that Not5 is needed in the cytoplasm for the co-translational assembly of RNA polymerase II. This stems from the importance of Not5 for the association of the R2TP Hsp90 co-chaperone with polysomes translating RPB1 mRNA to protect newly synthesized Rpb1 from aggregation. Hence taken together our results show that Not5 interconnects translation and transcription

10th Francophone Yeast Meeting 'Levures, Modèles & Outils'

Meeting reportBetween the first and tenth editions of this biannual rendezvous, yeasters were faced with an extraordinary revolution: Saccharomyces cerevisiae was indeed the first eukaryotic genome completely sequenced and released to the community, reviving its enormous potential for understanding life and opening the door to new approaches in biology. This revolution was immediately followed by ambitious programmes for sequencing the genomes of its distant cousins, e.g. the ‘Genolevures’ program held by a consortium of seven French laboratories, with the first aim of deciphering the different mechanisms of eukaryotic genome evolution over long periods of time. Taking advantage of the knowledge of these new genomes, researchers can now tackle much more easily relevant physiological studies in very different and distant yeasts, the diversity of which has once again been attractively represented at this 10th meeting. In addition to S. cerevisiae and Saccharomyces pombe still highly valued by researchers, many oral and written communications attested to the growing interest of our community for these ‘less-conventional’ yeasts. This explains why, in parallel with this genomic revolution, a mutation naturally occurred in the title of this meeting, with the recent emergence of three ‘s’ and subsequent transition from ‘Levure, Mode`le & Outil’ to ‘‘Levures, Mode`les & Outils’. This latest edition of LMO therefore brought together 250 participants from fields that rarely interact in order to discuss the current status of their research interests. This massive participation certified that our community remains very active, but also that it is particularly fond of this appointment. It offers a panoramic view of the extraordinary potential of yeasts, that continue to be driving forces in many fields of biology, from basic research to biotechnological challenges in coming years. Topics therefore ranged from a convincing demonstration of the central role of the mediator complex in RNA-polymerase II-dependent transcription to the engineering of new yeast strains to meet emerging energy problems that humanity will have to face in the near future. This report highlights some of the latest findings presented at this meeting, where two of the sessions were held in memory of renowned French colleagues Barbara Winsor and Pierre Thuriaux, who sadly passed away on the eve of retirement. 1. Beverages and energy: from ancestral use of yeasts to cutting edge biotechnological challenges2. Evolution and the genomics revolution3. Traffic and organelles: focus on mitochondrial ATP synthase4. Let’s travel into the nucleus: from DNA organisation to transcription5. RNA life, from synthesis to translation6. It’s all about control: key elements in cell cycle progression and establishment of cell polarit

Rpb4 interacts with Not5 and the cytoplasmic functions of Rpb4 require Not5.

<p><b>A</b>. Serial dilutions of exponentially growing reporter cells expressing LexA-Rpb4 as a bait, and the indicated proteins fused to B42 as preys, were spotted either on medium selective for the plasmids (left panel +L) or selective for the plasmids and indicative of an interaction between bait and prey (right panel -L). <b>B</b>. Serial dilutions of exponentially growing cells from the indicated strains were spotted on plates and left to grow for several days at 30°C. <b>C</b> and <b>D</b>. Fractions from 7–47% sucrose gradients of extracts from wild-type or mutant strains expressing the indicated Tap-tagged (TT) proteins were precipitated with TCA and analyzed by western blotting with PAP antibodies. The positions of 40S, 60S, 80S and polysomes are indicated under the blots. The numbers of the gradient fractions tested or the total extract (TE) are indicated at the top. The polysome profiles for these experiments are available in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004569#pgen.1004569.s015" target="_blank">Fig. S15</a> along with a typical distribution of a ribosomal protein (Rps3) in the wt and <i>not5Δ</i> gradients. Rpb4-TT (<b>E</b>) or Not1-TT (<b>F</b>) were immunoprecipitated from extracts of wild-type or mutant cells expressing HA-tagged Prt1. Wild-type cells expressing untagged Rpb4 or Not1 were used as a control. Similar negative controls were obtained with <i>not5Δ</i> cells not expressing any Tap-tagged protein (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004569#pgen.1004569.s016" target="_blank">Fig. S16</a>). The immunoblots were developed using anti-CBP or HA antibodies. <b>G</b> and <b>H</b>. Wild-type and <i>not5Δ</i> cells expressing Rpb4-TT (<b>G</b>) or the indicated (<b>H</b>) Not5 derivatives, were grown exponentially and stained with anti-CBP antibodies (upper panels) or DAPI (middle panels). The pictures were merged (lower panels) and the indicated section from wild-type (a) or <i>not5Δ</i> (b) was enlarged for better visualization. The localization of the Not5 derivatives is presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004569#pgen.1004569.s002" target="_blank">Fig. S2</a>.</p

Polymerase sub-complexes lacking Rpb1 accumulate in <i>not5Δ</i>.

<p><b>A</b> and <b>B</b>. Total extracts from cells expressing the indicated Tap-tagged (TT) polymerase subunits were separated on native gels (upper panels) or SDS-PAGE (lower panels) and analyzed by western blotting with anti- CBP antibodies. <b>C</b>. Rpb9-TT was purified by single step affinity and the purified proteins were analyzed on native gels (upper panels) or SDS-PAGE (lower panels) and western blotting with anti-CBP antibodies (left panel) or anti-Rpb1 antibodies (right panel). <b>D</b>. Total extracts from cells expressing Rpb11-TT were either untreated (-) or treated with DNase or RNase as indicated and separated by Native-PAGE, and analyzed by western blotting with PAP antibodies.</p

Presence of Rpb7 in polysomes or in the cytoplasm is not affected by Not5.

<p><b>A</b>. Wild-type or mutant cells expressing Tap-tagged Rpb7 as indicated were analyzed on sucrose gradients as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004569#pgen-1004569-g001" target="_blank">Fig. 1C</a>. The polysome profiles and protein loading for these experiments are available in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004569#pgen.1004569.s015" target="_blank">Fig. S15</a>. <b>B</b>. Wild-type and <i>not5Δ</i> cells expressing Rpb7-TT were grown exponentially and stained with anti-CBP antibodies or DAPI as for Fig. 1G. The merged pictures are displayed.</p