Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity

RNA silencing regulates gene expression through mRNA degradation, translation repression and chromatin remodelling. The fundamental engines of RNA silencing are RISC and RITS complexes, whose common components are 21-25 nt RNA and an Argonaute protein containing a PIWI domain of unknown function. The crystal structure of an archaeal Piwi protein (AfPiwi) is organised into two domains, one resembling the sugar-binding portion of the lac repressor and another with similarity to RNase H. Invariant residues and a coordinated metal ion lie in a pocket that surrounds the conserved C-terminus of the protein, defining a key functional region in the PIWI domain. Furthermore, two Asp residues, conserved in the majority of Argonaute sequences, align spatially with the catalytic Asp residues of RNase H-like catalytic sites, suggesting that in eukaryotic Argonaute proteins the RNase H-like domain may possess nuclease activity. The conserved region around the C-terminus of the PIWI domain, which is required for small interfering RNA (siRNA) binding to AfPiwi, may function as the receptor site for the obligatory 5' phosphate of siRNAs, thereby specifying the cleavage position of the target mRNA.     

Miles to go (mtgo) encodes FNDC3 proteins that interact with the chaperonin subunit CCT3 and are required for NMJ branching and growth in Drosophila

Analysis of mutants that affect formation and function of the Drosophila larval neuromuscular junction (NMJ) has provided valuable insight into genes required for neuronal branching and synaptic growth. We report that NMJ development in Drosophila requires both the Drosophila ortholog of FNDC3 genes; CG42389 (herein referred to as miles to go; mtgo), and CCT3, which encodes a chaperonin complex subunit. Loss of mtgo function causes late pupal lethality with most animals unable to escape the pupal case, while rare escapers exhibit an ataxic gait and reduced lifespan. NMJs in mtgo mutant larvae have dramatically reduced branching and growth and fewer synaptic boutons compared with control animals. Mutant larvae show normal locomotion but display an abnormal self-righting response and chemosensory deficits that suggest additional functions of mtgo within the nervous system. The pharate lethality in mtgo mutants can be rescued by both low-level pan- and neuronal-, but not muscle-specific expression of a mtgo transgene, supporting a neuronal-intrinsic requirement for mtgo in NMJ development. Mtgo encodes three similar proteins whose domain structure is most closely related to the vertebrate intracellular cytosolic membrane-anchored fibronectin type-III domain-containing protein 3 (FNDC3) protein family. Mtgo physically and genetically interacts with Drosophila CCT3, which encodes a subunit of the TRiC/CCT chaperonin complex required for maturation of actin, tubulin and other substrates. Drosophila larvae heterozygous for a mutation in CCT3 that reduces binding between CCT3 and MTGO also show abnormal NMJ development similar to that observed in mtgo null mutants. Hence, the intracellular FNDC3-ortholog MTGO and CCT3 can form a macromolecular complex, and are both required for NMJ development in Drosophila.     

Chromator is required for proper microtubule spindle formation and mitosis in Drosophila

The chromodomain protein, Chromator, has been shown to have multiple functions that include regulation of chromatin structure as well as coordination of muscle remodeling during metamorphosis depending on the developmental context. In this study we show that mitotic neuroblasts from brain squash preparations from larvae heteroallelic for the two Chromator loss-of-function alleles Chro⁷¹ and Chro⁶¹² have severe microtubule spindle and chromosome segregation defects that were associated with a reduction in brain size. The microtubule spindles formed were incomplete, unfocused, and/or without clear spindle poles and at anaphase chromosomes were lagging and scattered. Time-lapse analysis of mitosis in S2 cells depleted of Chromator by RNAi treatment suggested that the lagging and scattered chromosome phenotypes were caused by incomplete alignment of chromosomes at the metaphase plate, possibly due to a defective spindle-assembly checkpoint, as well as of frayed and unstable microtubule spindles during anaphase. Expression of full-length Chromator transgenes under endogenous promoter control restored both microtubule spindle morphology as well as brain size strongly indicating that the observed mutant defects were directly attributable to lack of Chromator function.     

Cell surface proteins in the early embryogenesis of Pleurodeles waltlii

Surface proteins in the first embryonic stages (8–32 cells, morula, blastula, early and late gastrula) of Pleurodeles waltlii were selectively labelled by ¹²⁵I using lactoperoxidase and glucose/glucose oxidase. Iodination was effected either on non-dissociated embryos or after their dissociation with EDTA. On the outer surface of non-dissociated embryos the two-dimensional electrophoresis revealed only three groups of ¹²⁵I-labelled proteins which did not change during all studied stages. Quite different results were obtained with the cells of dissociated embryos. In addition to the iodinated proteins of the embryonic outer surface seven major iodinated proteins were identified. These proteins originate from the regions of cell-cell contacts in intact embryo. Their two-dimensional pattern in dissociated cells changes between stages 8–32 cells and morula. The next important difference was observed during gastrulation, which corresponds in Pleurodeles waltlii to the first morphogenetic movements. Therefore the outside and inside cell surfaces of embryo are different already at stage 8–32 cells (and probably earlier), before the first step of morphogenesis. The changes of cell surface proteins at early embryonal development take place inside the embryo, in the regions of cell-cell interactions.     

Developmental regulation of glial cell phagocytic function during Drosophila embryogenesis

The proper removal of superfluous neurons through apoptosis and subsequent phagocytosis is essential for normal development of the central nervous system (CNS). During Drosophila embryogenesis, a large number of apoptotic neurons are efficiently engulfed and degraded by phagocytic glia. Here we demonstrate that glial proficiency to phagocytose relies on expression of phagocytic receptors for apoptotic cells, SIMU and DRPR. Moreover, we reveal that the phagocytic ability of embryonic glia is established as part of a developmental program responsible for glial cell fate determination and is not triggered by apoptosis per se. Explicitly, we provide evidence for a critical role of the major regulators of glial identity, gcm and repo, in controlling glial phagocytic function through regulation of SIMU and DRPR specific expression. Taken together, our study uncovers molecular mechanisms essential for establishment of embryonic glia as primary phagocytes during CNS development.     

The small ubiquitin-like modifier (SUMO) is essential in cell cycle regulation in Trypanosoma brucei

SUMO, a reversible post-translational protein modifier, plays important roles in many processes of higher eukaryotic cell life. Although SUMO has been identified in many eukaryotes, SUMO and SUMO system are still unknown in some eukaryotic unicellular organisms, such as Trypanosoma brucei (T. brucei). In this study, only one SUMO homologue (TbSUMO) was identified in T. brucei. Expression of TbSUMO was knocked down by using RNA interference technique in procyclic-form T. brucei. The growth of TbSUMO-deficient cells was significantly inhibited. TbSUMO-deficient cells were arrested in G₂/M phase accompanied with an obvious increase of 0N1K cells (zoids), and failed in chromosome segregation. These results indicate that TbSUMO is essential in cell cycle regulation, with one important role in mitosis. Meanwhile, the enrichment of zoids suggests the inhibition of mitosis does not prevent the cell division in procyclic-form T. brucei. HA-tagged TbSUMO was overexpressed in T. brucei and was shown to be localized to the nucleus through the whole cell cycle, further revealing its distinguished functions in nucleus. All these accumulated data imply that a SUMO system essential for regulating cell cycle progression might exist in the procyclic-form T. brucei.     

Drosophila RecQ5 is involved in proper progression of early spermatogenesis

RecQ5, a member of the conserved RecQ DNA helicase family, is required for the maintenance of genome stability. The human RECQL5 gene is expressed ubiquitously in almost all tissues, with strong expression in the testes (Shimamoto et al., 2000). However, it remains to be elucidated in which cells RecQ5 is expressed and how RecQ5 functions in the testes. In this present study we analyzed the expression of RecQ5 in Drosophila testes. The RecQ5 protein was specifically expressed in germline cells in larval, pupal, and adult testes. Drosophila RecQ5 was localized in nuclei of male germline stem cells, spermatogoniablasts, spermatogonia, and early spermatocytes. As growth of the early spermatocyte proceeded, the amount of RecQ5 increased in the nuclei. However, before maturation of the spermatocyte, the level of RecQ5 declined. Thus, RecQ5 expression was regulated. Furthermore, we compared recq5 mutant testes with the wild-type ones. The most conspicuous alterations were swelling of the apical region of and an increase in the number of spermatocytes in the recq5 testis, suggesting a relative accumulation of spermatocytes in the recq5 mutant testes. Therefore, Drosophila RecQ5 may contribute to the proper progression from germline stem cells to spermatocytes for maintenance of genome stability.     

Jak-STAT regulation of male germline stem cell establishment during Drosophila embryogenesis

Germline stem cells (GSCs) in Drosophila are descendants of primordial germ cells (PGCs) specified during embryogenesis. The precise timing of GSC establishment in the testis has not been determined, nor is it known whether mechanisms that control GSC maintenance in the adult are involved in GSC establishment. Here, we determine that PGCs in the developing male gonad first become GSCs at the embryo to larval transition. This coincides with formation of the embryonic hub; the critical signaling center that regulates adult GSC behavior within the stem cell microenvironment (niche). We find that the Jak-STAT signaling pathway is activated in a subset of PGCs that associate with the newly-formed embryonic hub. These PGCs express GSC markers and function like GSCs, while PGCs that do not associate with the hub begin to differentiate. In the absence of Jak-STAT activation, PGCs adjacent to the hub fail to exhibit the characteristics of GSCs, while ectopic activation of the Jak-STAT pathway prevents differentiation. These findings show that stem cell formation is closely linked to development of the stem cell niche, and suggest that Jak-STAT signaling is required for initial establishment of the GSC population in developing testes.     

Autophagy takes flight in Drosophila

Drosophila has been shown to be a powerful model to study autophagy, whose regulation involves a core machinery consisting of Atg proteins and upstream signaling regulators similar to those in yeast and mammals. The conserved role in degrading proteins and organelles gives autophagy an important function in coordinating several cellular processes as well as in a number of pathological conditions. This review summarizes key studies in Drosophila autophagy research and discusses potential questions that may lead to better understanding of the roles and regulation of autophagy in higher eukaryotes.     

Drosophila SMN complex proteins Gemin2, Gemin3, and Gemin5 are components of U bodies

Uridine-rich small nuclear ribonucleoproteins (U snRNPs) play key roles in pre-mRNA processing in the nucleus. The assembly of most U snRNPs takes place in the cytoplasm and is facilitated by the survival motor neuron (SMN) complex. Discrete cytoplasmic RNA granules called U bodies have been proposed to be specific sites for snRNP assembly because they contain U snRNPs and SMN. U bodies invariably associate with P bodies, which are involved in mRNA decay and translational control. However, it remains unknown whether other SMN complex proteins also localise to U bodies. In Drosophila there are four SMN complex proteins, namely SMN, Gemin2/CG10419, Gemin3 and Gemin5/Rigor mortis. Drosophila Gemin3 was originally identified as the Drosophila orthologue of human and yeast Dhh1, a component of P bodies. Through an in silico analysis of the DEAD-box RNA helicases we confirmed that Gemin3 is the bona fide Drosophila orthologue of vertebrate Gemin3 whereas the Drosophila orthologue of Dhh1 is Me31B. We then made use of the Drosophila egg chamber as a model system to study the subcellular distribution of the Gemin proteins as well as Me31B. Our cytological investigations show that Gemin2, Gemin3 and Gemin5 colocalise with SMN in U bodies. Although they are excluded from P bodies, as components of U bodies, Gemin2, Gemin3 and Gemin5 are consistently found associated with P bodies, wherein Me31B resides. In addition to a role in snRNP biogenesis, SMN complexes residing in U bodies may also be involved in mRNP assembly and/or transport.     

Investigations on ecdysteroids and juvenile hormones and on morphological aspects during early embryogenesis in the ovoviviparous cockroach Nauphoeta cinerea

During early embryogenesis, which is from ovulation (day 0) until dorsal closure (day 19), the quantity of free and conjugated ecdysteroids in the egg cases, as measured by radioimmunoassay (RIA), increases. Thin-layer chromatography (TLC) and high-performance-liquid-chromatography (HPLC) analyses combined with RIA suggest that 20-hydroxy-ecdysone is the predominant ecdysteroid. Hydrolysis of the highly polar products of day-0 and day-17 egg cases by Helix pomatia enzymes indicates the presence of some conjugates of 20-hydroxy-ecdysone, hydrolyzable under these conditions. However, important quantities of RIA-reactive highly polar products are not hydrolyzed particularly in day-17 egg cases. These results demonstrate that the highly polar products of day-0 egg cases are qualitatively as well as quantitatively different from the highly polar products of day-17 egg cases. Morphological investigations show that the peak of 20-hydroxy-ecdysone at the time of the dorsal closure coincides with the synthesis of an embryonic cuticle. Using the Galleria wax test only traces, or no juvenile hormone activity could be detected in embryos during the entire period of early embryonic development. Morphological investigations of the brood sac suggest that this organ is very important to facilitate the initial uptake of water into the eggs. Thereafter the embryos can develop independently of the female when kept in a humid environment.     

mRNA diffusion explains protein gradients in Drosophila early development

We propose a new model describing the production and the establishment of the stable gradient of the Bicoid protein along the antero-posterior axis of the embryo of Drosophila. In this model, we consider that bicoid mRNA diffuses along the antero-posterior axis of the embryo and the protein is produced in the ribosomes localized near the syncytial nuclei. Bicoid protein stays localized near the syncytial nuclei as observed in experiments. We calibrate the parameters of the mathematical model with experimental data taken during the cleavage stages 11-14 of the developing embryo of Drosophila. We obtain good agreement between the experimental and the model gradients, with relative errors in the range 5-8%. The inferred diffusion coefficient of bicoid mRNA is in the range , in agreement with the theoretical predictions and experimental measurements for the diffusion of macromolecules in the cytoplasm. We show that the model based on the mRNA diffusion hypothesis is consistent with the known observational data, supporting the recent experimental findings of the gradient of bicoid mRNA in Drosophila [Spirov et al. (2009). Development 136, 605-614].     

Arabinogalactan proteins are essential in somatic embryogenesis of Daucus carota L.

Daucus carota L. cell lines secrete a characteristic set of arabinogalactan proteins (AGPs) into the medium. The composition of this set of AGPs changes with the age of the culture, as can be determined by crossed electrophoresis with the specific AGP-binding agent, β-glucosyl Yariv reagent. Addition of AGPs isolated from the medium of a non-embryogenic cell line to an expiant culture initiated the development of the culture to a non-embryogenic cell line. Without addition of AGPs or with addition of carrot-seed AGPs an embryogenic cell line was established. Three-month-old embryogenic cell lines usually contain less than 30% of dense, highly cytoplasmic cells, i.e. the embryogenic cells, but when carrot-seed AGPs were added this percentage increased to 80%. Addition of carrot-seed AGPs to a two-year-old, non-embryogenic cell line resulted in the re-induction of embryogenic potential. These results show that specific AGPs are essential in somatic embryogenesis and are able to direct development of cells.     

Cleavage of the Drosophila screw prodomain is critical for a dynamic BMP morphogen gradient in embryogenesis

Dorsoventral patterning of the Drosophila embryo is regulated by graded distribution of bone morphogenetic proteins (BMPs) composed of two ligands, decapentaplegic (Dpp) a BMP2/4 ortholog and screw (Scw) a BMP5/6/7/8 family member. scw^E1 encodes an unusual allele that was isolated as a dominant enhancer of partial loss-of-function mutations in dpp. However, the molecular mechanisms that underlie this genetic interaction remain to be addressed. Here we show that scw^E1 contains a mutation at the furin cleavage site within the prodomain that is crucial for ligand production. Furthermore, our data show that Scw^E1 preferentially forms heterodimers with Dpp rather than homotypic dimers, providing a possible explanation for the dominant negative phenotype of scw^E1 alleles. The unprocessed prodomain of Scw^E1 remains in a complex with the Dpp:Scw heterodimer, and thus could interfere with interaction of the ligand with the extracellular matrix, or the kinetics of processing/secretion of the ligand in vivo. These data reveal novel mechanisms by which post-translational regulation of Scw can modulate Dpp signaling activity.     

New yeast/E. coli/Drosophila triple shuttle vectors for efficient generation of Drosophila P element transformation constructs

We have generated a set of novel triple shuttle vectors that facilitate the construction of Drosophila-P-element transformations vectors. These YED-vectors allow the insertion of any kind of sequence at any chosen position due to the presence of a yeast casette which ensures replication and allows for homologous recombination in Saccharomyces cerevisiae. As a proof of principle we generated several reporter constructs and tested them in transgenic flies for expression and correct subcellular localization. YED-vectors can be used for many purposes including promoter analysis or the expression of tagged or truncated proteins. Thus, time-consuming conventional restriction site based multi-step cloning procedures can be circumvented by using the new YED-vectors. The new set of triple shuttle vectors will be highly beneficial for the rapid construction of complex Drosophila transformation plasmids.     

Drosophila Syntrophins are involved in locomotion and regulation of synaptic morphology

Syntrophin components of the dystrophin glycoprotein complex (DGC) feature multiple protein interaction domains that may act in molecular scaffolding, recruiting signaling proteins to membranes and the DGC. Drosophila Syntrophin-1 (Syn1) and Syntrophin-2 (Syn2) are counterparts of human α1/β1/β2-syntrophins and γ1/γ2-syntrophins, respectively. α1/β1/β2-syntrophins are well documented, while little is known about γ1/γ2-syntrophins. Here, we performed immunohistochemical analyses with a specific antibody to Syn2 and demonstrated predominant expression in the larval and adult central nervous system. To investigate the in vivo functions of Syn2, we have generated Drosophila Syn2 deficiency mutants. Although the Syn2 mutants exhibit no overt phenotype, the combination of Syn1 knockdown and Syn2³⁷ mutation dramatically shortened life span, synergistically reduced locomotion ability and synergistically enhanced overgrowth of neuromuscular junctions in N-ethylmaleimide sensitive factor 2 mutants. From these data we conclude that Syn1 and Syn2 are required for locomotion and are involved in regulation of synaptic morphology. In addition, the two syntrophins can at least partially compensate for each other's functions.