Alain Camasses

The Nucle(ol)ar Tif6p and Efl1p Are Required for a Late Cytoplasmic Step of Ribosome Synthesis

Deletion of elongation factor-like 1 (Efl1p), a cytoplasmic GTPase homologous to the ribosomal translocases EF-G/EF-2, results in nucle(ol)ar pre-rRNA processing and pre-60S subunits export defects. Efl1p interacts genetically with Tif6p, a nucle(ol)ar protein stably associated with pre-60S subunits and required for their synthesis and nuclear exit. In the absence of Efl1p, 50% of Tif6p is relocated to the cytoplasm. In vitro, the GTPase activity of Efl1p is stimulated by 60S, and Efl1p promotes the dissociation of Tif6p-60S complexes. We propose that Tif6p binds to the pre-60S subunits in the nucle(ol)us and escorts them to the cytoplasm where the GTPase activity of Efl1p triggers a late structural rearrangement, which facilitates the release of Tif6p and its recycling to the nucle(ol)us.

Amphioxus FGF signaling predicts the acquisition of vertebrate morphological traits

FGF signaling is one of the few cell–cell signaling pathways conserved among all metazoans. The diversity of FGF gene content among different phyla suggests that evolution of FGF signaling may have participated in generating the current variety of animal forms. Vertebrates possess the greatest number of FGF genes, the functional evolution of which may have been implicated in the acquisition of vertebrate-specific morphological traits. In this study, we have investigated the roles of the FGF signal during embryogenesis of the cephalochordate amphioxus, the best proxy for the chordate ancestor. We first isolate the full FGF gene complement and determine the evolutionary relationships between amphioxus and vertebrate FGFs via phylogenetic and synteny conservation analysis. Using pharmacological treatments, we inhibit the FGF signaling pathway in amphioxus embryos in different time windows. Our results show that the requirement for FGF signaling during gastrulation is a conserved character among chordates, whereas this signal is not necessary for neural induction in amphioxus, in contrast to what is known in vertebrates. We also show that FGF signal, acting through the MAPK pathway, is necessary for the formation of the most anterior somites in amphioxus, whereas more posterior somite formation is not FGF-dependent. This result leads us to propose that modification of the FGF signal function in the anterior paraxial mesoderm in an amphioxus-like vertebrate ancestor might have contributed to the loss of segmentation in the preotic paraxial mesoderm of the vertebrate head.

Interactions within the Yeast Sm Core Complex: from Proteins to Amino Acids

ABSTRACT Sm core proteins play an essential role in the formation of small nuclear ribonucleoprotein particles (snRNPs) by binding to small nuclear RNAs and participating in a network of protein interactions. The two-hybrid system was used to identify SmE interacting proteins and to test for interactions between all pairwise combinations of yeast Sm proteins. We observed interactions between SmB and SmD3, SmE and SmF, and SmE and SmG. For these interactions, a direct biochemical assay confirmed the validity of the results obtained in vivo. To map the protein-protein interaction surface of Sm proteins, we generated a library of SmE mutants and investigated their ability to interact with SmF and/or SmG proteins in the two-hybrid system. Several classes of mutants were observed: some mutants are unable to interact with either SmF or SmG proteins, some interact with SmG but not with SmF, while others interact moderately with SmF but not with SmG. Our mutational analysis of yeast SmE protein shows that conserved hydrophobic residues are essential for interactions with SmF and SmG as well as for viability. Surprisingly, we observed that other evolutionarily conserved positions are tolerant to mutations, with substitutions affecting binding to SmF and SmG only mildly and conferring a wild-type growth phenotype.

The cell proliferation antigen Ki-67 organises heterochromatin

Antigen Ki-67 is a nuclear protein expressed in proliferating mammalian cells. It is widely used in cancer histopathology but its functions remain unclear. Here, we show that Ki-67 controls heterochromatin organisation. Altering Ki-67 expression levels did not significantly affect cell proliferation in vivo. Ki-67 mutant mice developed normally and cells lacking Ki-67 proliferated efficiently. Conversely, upregulation of Ki-67 expression in differentiated tissues did not prevent cell cycle arrest. Ki-67 interactors included proteins involved in nucleolar processes and chromatin regulators. Ki-67 depletion disrupted nucleologenesis but did not inhibit pre-rRNA processing. In contrast, it altered gene expression. Ki-67 silencing also had wide-ranging effects on chromatin organisation, disrupting heterochromatin compaction and long-range genomic interactions. Trimethylation of histone H3K9 and H4K20 was relocalised within the nucleus. Finally, overexpression of human or Xenopus Ki-67 induced ectopic heterochromatin formation. Altogether, our results suggest that Ki-67 expression in proliferating cells spatially organises heterochromatin, thereby controlling gene expression. DOI: http://dx.doi.org/10.7554/eLife.13722.001

RPK1, an essential yeast protein kinase involved in the regulation of the onset of mitosis, shows homology to mammalian dual-specificity kinases

We report here the sequence of RPK1 (for Regulatory cell Proliferation Kinase), a new Saccharomyces cerevisiae gene coding for a protein with sequence similarities to serine/threonine protein kinases. The protein sequence of 764 amino acids includes an amino-terminal domain (residues 1–410), which may be involved in regulation of the kinase domain (residues 411–764). The catalytic domain of Rpkl is not closely related to other known yeast protein kinases but exhibits strong homology to a newly discovered group of mammalian kinases (PYT, TTK, esk) with serine/threonine/tyrosine kinase activity. Null alleles of RPK1 are lethal and thus this gene belongs to the small group of yeast protein kinase genes that are essential for cell growth. In addition, eliminating the expression of RPK1 gives rise to the accumulation of non-viable cells with less than a 1 N DNA content suggesting that cells proceed into mitosis without completion of DNA synthesis. Therefore, the Rpkt kinase may function in a checkpoint control which couples DNA replication to mitosis. The level of the RPK1 transcript is extremely low and constant throughout the mitotic cycle. However it is regulated during cellular differentiation, being decreased in α-factor-treated a cells and increased late in meiosis in a/α diploids. Taken together, our results suggest that Rpk1 is involved in a pathway that coordinates cell proliferation and differentiation.

Dynamics of Nucleosome Positioning Maturation following Genomic Replication

Summary Chromatin is thought to carry epigenetic information from one generation to the next, although it is unclear how such information survives the disruptions of nucleosomal architecture occurring during genomic replication. Here, we measure a key aspect of chromatin structure dynamics during replication—how rapidly nucleosome positions are established on the newly replicated daughter genomes. By isolating newly synthesized DNA marked with 5-ethynyl-2′-deoxyuridine (EdU), we characterize nucleosome positions on both daughter genomes of S. cerevisiae during chromatin maturation. We find that nucleosomes rapidly adopt their mid-log positions at highly transcribed genes, which is consistent with a role for transcription in positioning nucleosomes in vivo. Additionally, experiments in hir1Δ mutants reveal a role for HIR in nucleosome spacing. We also characterized nucleosome positions on the leading and lagging strands, uncovering differences in chromatin maturation dynamics at hundreds of genes. Our data define the maturation dynamics of newly replicated chromatin and support a role for transcription in sculpting the chromatin template.

Actors of the tyrosine kinase receptor downstream signaling pathways in amphioxus

SUMMARY One of the major goals of evo‐developmentalists is to understand how the genetic mechanisms controlling embryonic development have evolved to create the current diversity of bodyplans that we encounter in the animal kingdom. Tyrosine kinase receptors (RTKs) are transmembrane receptors present in all metazoans known to control several developmental processes. They act via the activation of various cytoplasmic signaling cascades, including the mitogen‐activated protein kinase (MAPK), the PI3K/Akt, and the phospholipase C‐γ (PLCγ)/protein kinase C (PKC) pathways. In order to address the evolution of these three pathways and their involvement during embryogenesis in chordates, we took advantage of the complete genome sequencing of a key evolutionarily positioned species, the cephalochordate amphioxus, and searched for the complete gene set of the three signaling pathways. We found that the amphioxus genome contains all of the most important modules of the RTK‐activated cascades, and looked at the embryonic expression of two genes selected from each cascade. Our data suggest that although the PI3K/Akt pathway may have ubiquitous functions, the MAPK and the PLCγ/PKC cascades may play specific roles in amphioxus development. Together with data known in vertebrates, the expression pattern of PKC in amphioxus suggests that the PLCγ/PKC cascade was implicated in neural development in the ancestor of all chordates.

Initiation of DNA replication requires actin dynamics and formin activity

Nuclear actin regulates transcriptional programmes in a manner dependent on its levels and polymerisation state. This dynamics is determined by the balance of nucleocytoplasmic shuttling, formin‐ and redox‐dependent filament polymerisation. Here, using Xenopus egg extracts and human somatic cells, we show that actin dynamics and formins are essential for DNA replication. In proliferating cells, formin inhibition abolishes nuclear transport and initiation of DNA replication, as well as general transcription. In replicating nuclei from transcriptionally silent Xenopus egg extracts, we identified numerous actin regulators, and disruption of actin dynamics abrogates nuclear transport, preventing NLS (nuclear localisation signal)‐cargo release from RanGTP–importin complexes. Nuclear formin activity is further required to promote loading of cyclin‐dependent kinase (CDK) and proliferating cell nuclear antigen (PCNA) onto chromatin, as well as initiation and elongation of DNA replication. Therefore, actin dynamics and formins control DNA replication by multiple direct and indirect mechanisms.