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Trimethylguanosine synthase 1 (TGS1) is a highly conserved enzyme that converts the 5′-monomethylguanosine cap of small nuclear RNAs (snRNAs) to a trimethylguanosine cap. Here, we show that loss of TGS1 in Caenorhabditis elegans, Drosophila melanogaster and Danio rerio results in neurological phenotypes similar to those caused by survival motor neuron (SMN) deficiency. Importantly, expression of human TGS1 ameliorates the SMN-dependent neurological phenotypes in both flies and worms, revealing that TGS1 can partly counteract the effects of SMN deficiency. TGS1 loss in HeLa cells leads to the accumulation of immature U2 and U4atac snRNAs with long 3′ tails that are often uridylated. snRNAs with defective 3′ terminations also accumulate in Drosophila Tgs1 mutants. Consistent with defective snRNA maturation, TGS1 and SMN mutant cells also exhibit partially overlapping transcriptome alterations that include aberrantly spliced and readthrough transcripts. Together, these results identify a neuroprotective function for TGS1 and reinforce the view that defective snRNA maturation affects neuronal viability and function.

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Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimethylguanosine cap on several RNAs, including snRNAs and telomerase RNA. Previous studies have shown that TGS1 binds the Survival Motor Neuron (SMN) protein, whose deficiency causes spinal muscular atrophy (SMA). In addition, TGS1 depletion results in increased hTR levels and telomere elongation in human cells. Here, we analyzed the roles of theDrosophilaorthologs of the humanTGS1andSMNgenes. We show that theDrosophilaTGS1 protein (dTgs1) physically interacts with all subunits of theDrosophilaSmn complex (Smn, Gem2, Gem3, Gem4 and Gem5), and that a humanTGS1transgene rescues the mutant phenotype caused bydTgs1loss. We demonstrate that bothdTgs1andSmnare required for viability of retinal progenitor cells and that downregulation of these genes leads to a reduced eye size. Importantly, overexpression ofdTgs1partially rescues the eye defects caused by Smn depletion, and vice versa. These results suggest that theDrosophilaeye model can be exploited for screens aimed at the identification of genes and drugs that modify the phenotypes elicited by Tgs1 and Smn deficiency. These modifiers could help to devise new therapies for SMA and diseases caused by telomerase insufficiency.

Morgana (Mora, also known as CHORD in flies) and its mammalian homologue, called CHORDC1 or CHP1, is a highly conserved cysteine and histidine-rich domain (CHORD)-containing protein that has been proposed to function as an Hsp90 co-chaperone. Morgana deregulation promotes carcinogenesis in both mice and humans while, in Drosophila, loss of mora causes lethality and a complex mitotic phenotype that is rescued by a human morgana transgene. Here, we show that Drosophila Mora localises to mitotic spindles and co-purifies with the Hsp90–R2TP–TTT supercomplex and with additional well-known Hsp90 co-chaperones. Acute inhibition of Mora function in the early embryo results in a dramatic reduction in centrosomal microtubule stability, leading to small spindles nucleated from mitotic chromatin. Purified Mora binds to microtubules directly and promotes microtubule polymerisation in vitro, suggesting that Mora directly regulates spindle dynamics independently of its Hsp90 co-chaperone role., Summary: Morgana, a Drosophila homologue of an oncogene, physically interacts with Hsp90 complexes and has an independent function in regulating microtubule polymerisation, both in vitro and in vivo.

Trimethylguanosine synthase 1 (TGS1) is a highly conserved enzyme that converts the 5’ mono-methylguanosine cap of snRNAs to a trimethylguanosine cap. Here, we show that loss of TGS1 in C. elegans, D. melanogaster and D. rerio results in neurological phenotypes similar to those caused by Survival Motor Neuron (SMN) deficiency. Importantly, expression of human TGS1 ameliorates the SMN-dependent neurological phenotypes in both flies and worms, revealing that TGS1 can partly counteract the effects of SMN deficiency. TGS1 loss in HeLa cells leads to the accumulation of immature U2 and U4atac snRNAs with long 3’ tails that are often uridylated. snRNAs with defective 3’ terminations also accumulate in Drosophila Tgs1 mutants. Consistent with defective snRNA maturation, TGS1 and SMN mutant cells also exhibit partially overlapping transcriptome alterations that include aberrantly spliced and readthrough transcripts. Together, these results identify a neuroprotective function for TGS1 and reinforce the view that defective snRNA maturation affects neuronal viability and function.

Trimethylguanosine synthase 1 (TGS1) is a conserved enzyme that mediates formation of the trimethylguanosine cap on several RNAs, including snRNAs and telomerase RNA. Previous studies have shown that TGS1 binds the Survival Motor Neuron (SMN) protein, whose deficiency causes spinal muscular atrophy (SMA). Here, we analyzed the roles of the Drosophila orthologs of the human TGS1 and SMN genes. We show that the Drosophila TGS1 protein (dTgs1) physically interacts with all subunits of the Drosophila Smn complex (Smn, Gem2, Gem3, Gem4 and Gem5), and that a human TGS1 transgene rescues the mutant phenotype caused by dTgs1 loss. We demonstrate that both dTgs1 and Smn are required for viability of retinal progenitor cells and that downregulation of these genes leads to a reduced eye size. Importantly, overexpression of dTgs1 partially rescues the eye defects caused by Smn depletion, and vice versa. These results suggest that the Drosophila eye model can be exploited for screens aimed at the identification of genes and drugs that modify the phenotypes elicited by Tgs1 and Smn deficiency. These modifiers could help to understand the molecular mechanisms underlying SMA pathogenesis and devise new therapies for this genetic disease., Author summary We explored the functional relationships between TGS1 and SMN using Drosophila as model organism. TGS1 is an enzyme that modifies the structure of the 5’-end of several RNAs, including telomerase RNA and the small nuclear RNAs (snRNAs) that are required for messenger RNA maturation. The SMN protein regulates snRNAs biogenesis and mutations in human SMN cause Spinal Muscular Atrophy (SMA), a devastating disorder characterized by neurodegeneration, progressive paralysis and death. We show that mutations in the Drosophila TGS1 (dTgs1) gene cause lethality, which is rescued by a human TGS1 transgene. We also show that the dTgs1 protein physically interacts with all subunits of the Smn complex, and that downregulation of either dTgs1 or Smn leads to a reduced Drosophila eye size. Notably, overexpression of dTgs1 partially rescues the eye defects caused by Smn knockdown, and vice versa, indicating that these genes cooperate in eye development. These results suggest that the eye model can be exploited for screens aimed at detection of chemical and genetic modifiers of the eye mutant phenotype elicited by dTgs1 and Smn deficiency, providing new clues about SMA pathogenesis and potential therapies.

Many genes are required for the assembly of the mitotic apparatus and for proper chromosome behavior during mitosis and meiosis. A fruitful approach to elucidate the mechanisms underlying cell division is the accurate phenotypic characterization of mutations in these genes. Here, we report the identification and characterization of diamond (dind), an essential Drosophila gene required both for mitosis of larval brain cells and for male meiosis. Larvae homozygous for any of the five EMS-induced mutations die in the third-instar stage and exhibit multiple mitotic defects. Mutant brain cells exhibit poorly condensed chromosomes and frequent chromosome breaks and rearrangements; they also show centriole fragmentation, disorganized mitotic spindles, defective chromosome segregation, endoreduplicated metaphases, and hyperploid and polyploid cells. Comparable phenotypes occur in mutant spermatogonia and spermatocytes. The dind gene encodes a non-conserved protein with no known functional motifs. Although the Dind protein exhibits a rather diffuse localization in both interphase and mitotic cells, fractionation experiments indicate that some Dind is tightly associated with the chromatin. Collectively, these results suggest that loss of Dind affects chromatin organization leading to defects in chromosome condensation and integrity, which in turn affect centriole stability and spindle assembly. However, our results do not exclude the possibility that Dind directly affects some behaviors of the spindle and centrosomes.

SummaryMitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers [1, 2]. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions [3, 4]. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies [5], but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the Tubulin chaperone complex, TCP-1/CCT, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs.

Crosses between Drosophila melanogaster females and Drosophila simulans males produce hybrid sons that die at the larval stage. This hybrid lethality is suppressed by loss-of-function mutations in the D. melanogaster Hybrid male rescue (Hmr) or in the D. simulans Lethal hybrid rescue (Lhr) genes. Previous studies have shown that Hmr and Lhr interact with heterochromatin proteins and suppress expression of transposable elements within D. melanogaster. It also has been proposed that Hmr and Lhr function at the centromere. We examined mitotic divisions in larval brains from Hmr and Lhr single mutants and Hmr; Lhr double mutants in D. melanogaster. In none of the mutants did we observe defects in metaphase chromosome alignment or hyperploid cells, which are hallmarks of centromere or kinetochore dysfunction. In addition, we found that Hmr-HA and Lhr-HA do not colocalize with centromeres either during interphase or mitotic division. However, all mutants displayed anaphase bridges and chromosome aberrations resulting from the breakage of these bridges, predominantly at the euchromatin–heterochromatin junction. The few dividing cells present in hybrid males showed fuzzy and irregularly condensed chromosomes with unresolved sister chromatids. Despite this defect in condensation, chromosomes in hybrids managed to align on the metaphase plate and undergo anaphase. We conclude that there is no evidence for a centromeric function of Hmr and Lhr within D. melanogaster nor for a centromere defect causing hybrid lethality. Instead, we find that Hmr and Lhr are required in D. melanogaster for detachment of sister chromatids during anaphase.

Crosses between Drosophila melanogaster females and Drosophila simulans males produce hybrid sons that die at the larval stage. This hybrid lethality is suppressed by loss-of-function mutations in the D. melanogaster Hybrid male rescue (Hmr) or in the D. simulans Lethal hybrid rescue (Lhr) genes. Previous studies have shown that Hmr and Lhr interact with heterochromatin proteins and suppress expression of transposable elements within D. melanogaster. It also has been proposed that Hmr and Lhr function at the centromere. We examined mitotic divisions in larval brains from Hmr and Lhr single mutants and Hmr; Lhr double mutants in D. melanogaster. In none of the mutants did we observe defects in metaphase chromosome alignment or hyperploid cells, which are hallmarks of centromere or kinetochore dysfunction. In addition, we found that Hmr-HA and Lhr-HA do not colocalize with centromeres either during interphase or mitotic division. However, all mutants displayed anaphase bridges and chromosome aberrations resulting from the breakage of these bridges, predominantly at the euchromatin–heterochromatin junction. The few dividing cells present in hybrid males showed fuzzy and irregularly condensed chromosomes with unresolved sister chromatids. Despite this defect in condensation, chromosomes in hybrids managed to align on the metaphase plate and undergo anaphase. We conclude that there is no evidence for a centromeric function of Hmr and Lhr within D. melanogaster nor for a centromere defect causing hybrid lethality. Instead, we find that Hmr and Lhr are required in D. melanogaster for detachment of sister chromatids during anaphase.

Is Emperor Nero’s demagogy still up to date? Is his fight against Senatorial Class a step towards modern "Democracy" or was the Senatorial Class a valid counter power able to balance the Emperor’s growing power? Can we call "nice leadership" the attempt to conquer the popular consensus through arts and music performances? Both Nero and the Italian Premier Silvio Berlusconi seem to prefer this to a pure political and social consensus: how efficient can this be? What has it to do with modern Democracy? The article looks provocatively into these subjects in order to participate in the debate about political leadership.

The existence of latent centromeres has been proposed as a possible explanation for the ectopic emergence of neocentromeres in humans. This hypothesis predicts an association between the position of neocentromeres and the position of ancient centromeres inactivated during karyotypic evolution. Human chromosomal region 15q24-26 is one of several hotspots where multiple cases of neocentromere emergence have been reported, and it harbors a high density of chromosome-specific duplicons, rearrangements of which have been implicated as a susceptibility factor for panic and phobic disorders with joint laxity. We investigated the evolutionary history of this region in primates and found that it contains the site of an ancestral centromere which became inactivated about 25 million years ago, after great apes/Old World monkeys diverged. This inactivation has followed a noncentromeric chromosomal fission of an ancestral chromosome which gave rise to phylogenetic chromosomes XIV and XV in human and great apes. Detailed mapping of the ancient centromere and two neocentromeres in 15q24-26 has established that the neocentromere domains map approximately 8 Mb proximal and 1.5 Mb distal of the ancestral centromeric region, but that all three map within 500 kb of duplicons, copies of which flank the centromere in Old World Monkey species. This suggests that the association between neocentromere and ancestral centromere position on this chromosome may be due to the persistence of recombinogenic duplications accrued within the ancient pericentromere, rather than the retention of “centromere-competent” sequences per se. The high frequency of neocentromere emergence in the 15q24-26 region and the high density of clinically important duplicons are, therefore, understandable in the light of the evolutionary history of this region.

Pol32 is an accessory subunit of the replicative DNA Polymerase delta and of the translesion Polymerase zeta. Pol32 is involved in DNA replication, recombination and repair. Pol32's participation in high-and low-fidelity processes, together with the phenotypes arising from its disruption, imply multiple roles for this subunit within eukaryotic cells, not all of which have been fully elucidated. Using pol32 null mutants and two partial loss-of-function alleles pol32(rd1) and pol32(rds) in Drosophila melanogaster, we show that Pol32 plays an essential role in promoting genome stability. Pol32 is essential to ensure DNA replication in early embryogenesis and it participates in the repair of mitotic chromosome breakage. In addition we found that pol32 mutantssuppress position effect variegation, suggesting a role for Pol32 in chromatin architecture.

Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of ?- and ?-Tubulin hetero-dimers. Tubulins undergo a series of protein folding and post-translational modifications in order to fulfill their functions. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. We recently described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies, but the molecular function of Mst remains unknown. Here, we use affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. We demonstrate that Mst associates stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. We show that RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, we demonstrate that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, our structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, our results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the Tubulin-folding processes required for proper assembly of spindle MTs.

Summary Centrosome abnormalities lead to genomic instability and are a common feature of many cancer cells. Here we show that mutations in morgana/chp-1 result in centrosome amplification and lethality in both Drosophila and mouse, and that the fly centrosome phenotype is fully rescued by the human ortholog of morgana . In mouse cells, morgana forms a complex with Hsp90 and ROCK I and II, and directly binds ROCK II. Morgana downregulation promotes the interaction between ROCK II and nucleophosmin (NPM), leading to an increased ROCK II kinase activity, which results in centrosome amplification. morgana +/− primary cells and mice display an increased susceptibility to neoplastic transformation. In addition, tumor tissue array histochemical analysis revealed that morgana is underexpressed in a large fraction of breast and lung human cancers. Thus, morgana/chp-1 appears to prevent both centrosome amplification and tumorigenesis.

Background Melusin is a muscle specific signaling protein, required for compensatory hypertrophy response in pressure-overloaded heart. The role of Melusin in heart function has been established both by loss and gain of function experiments in murine models. With the aim of verifying the hypothesis of a potential role of the Melusin encoding gene, ITGB1BP2, in the modification of the clinical phenotype of human cardiomyopathies, we screened the ITGB1BP2 gene looking for genetic variations possibly associated to the pathological phenotype in three selected groups of patients affected by hypertension and dilated or hypertrophic cardiomyopathy Methods We analyzed ITGB1BP2 by direct sequencing of the 11 coding exons and intron flanking sequences in 928 subjects, including 656 hypertensive or cardiopathic patients and 272 healthy individuals. Results Only three nucleotide variations were found in patients of three distinct families: a C>T missense substitution at position 37 of exon 1 causing an amino acid change from His-13 to Tyr in the protein primary sequence, a duplication (IVS6+12_18dupTTTTGAG) near the 5'donor splice site of intron 6, and a silent 843C>T substitution in exon 11. Conclusions The three variations of the ITGB1BP2 gene have been detected in families of patients affected either by hypertension or primary hypertrophic cardiomyopathy; however, a clear genotype/phenotype correlation was not evident. Preliminary functional results and bioinformatic analysis seem to exclude a role for IVS6+12_18dupTTTTGAG and 843C>T in affecting splicing mechanism. Our analysis revealed an extremely low number of variations in the ITGB1BP2 gene in nearly 1000 hypertensive/cardiopathic and healthy individuals, thus suggesting a high degree of conservation of the melusin gene within the populations analyzed.

We have investigated the molecular evolution of the gene coding for beta-defensin 3 (DEFB103) in 17 primate species including humans. Unlike the DEFB4 genes (coding for beta-defensin 2) [Boniotto, Tossi, Del Pero, Sgubin, Antcheva, Santon and Masters (2003) Genes Immun. 4, 251-257], DEFB103 shows a marked degree of conservation in humans, Great Apes and New and Old World monkeys. Only the Hylobates concolor defensin hcBD3 showed an amino acid variation Arg17-->Trp17 that could have a functional implication, as it disrupts an intramolecular salt bridge with Glu27, which locally decreases the charge and may favour dimerization in the human congener hBD3. This is thought to involve the formation of an intermolecular salt bridge between Glu28 and Lys32 on another monomer [Schibli, Hunter, Aseyev, Starner, Wiencek, McCray, Tack and Vogel (2002) J. Biol. Chem. 277, 8279-8289 ]. To test the role of dimerization in mediating biological activity, we synthesized hBD3, hcBD3 and an artificial peptide in which the Lys26-Glu27-Glu28 stretch was replaced by the equivalent Phe-Thr-Lys stretch from human beta-defensin 1 and we characterized their structure and anti-microbial activity. Although the structuring and dimerization of these peptides were found to differ significantly, this did not appear to affect markedly the anti-microbial potency, the broad spectrum of activity or the insensitivity of the anti-microbial action to the salinity of the medium.