Maria Furia

The moulting hormone ecdysone is able to recognize target elements composed of direct repeats.

In Drosophila melanogaster, three temporally distinct ecdysone-responsive puff sets, the so-called intermoult, early and late puffs, have been described on the salivary gland polytene chromosomes. We have analyzed in detail a DNA segment of the 3C polytene region, from which the originates one of the most prominent intermoult puffs, with the aim of identifying ecdysone response elements (EcREs). Here we report that two putative EcREs of identical sequence are located at this puff site. Interestingly, these elements display a novel structural feature, being composed of directly repeated half-sites. Our results show that the EcR/USP heterodimer known to constitute the ecdysone functional receptor complex is able to bind to and transactivate through target elements composed of directly repeated half-sites. In addition, we show that these elements are also able to bind efficiently USP alone, suggesting that USP and EcR/USP could compete for their binding to DNA.

Human dyskerin: beyond telomeres

Abstract Human dyskerin is an evolutively conserved protein that participates in diverse nuclear complexes: the H/ACA snoRNPs, that control ribosome biogenesis, RNA pseudouridylation, and stability of H/ACA snoRNAs; the scaRNPs, that control pseudouridylation of snRNAs; and the telomerase active holoenzyme, which safeguards telomere integrity. The biological importance of dyskerin is further outlined by the fact that its deficiency causes the X-linked dyskeratosis congenita disease, while its over-expression characterizes several types of cancers and has been proposed as prognostic marker. The role of dyskerin in telomere maintenance has widely been discussed, while its functions as H/ACA sno/scaRNP component has been so far mostly overlooked and represent the main goal of this review. Here we summarize how increasing evidence indicates that the snoRNA/microRNA pathways can be interlaced, and that dyskerin-dependent RNA pseudouridylation represents a flexible mechanism able to modulate RNA function in different ways, including modulation of splicing, change of mRNA coding properties, and selective regulation of IRES-dependent translation. We also propose a speculative model that suggests that the dynamics of pre-assembly and nuclear import of H/ACA RNPs are crucial regulatory steps that can be finely controlled in the cytoplasm in response to developmental, differentiative and stress stimuli.

The role of the BR-C locus on the expression of genes located at the ecdysone-regulated 3C puff of Drosophila melanogaster

During the third larval instar, the steroid moulting hormone ecdysone activates three temporally distinct puff sets on the D. melanogaster salivary gland polytene chromosome: the so-called intermoult, early and late puffs. Hormonal regulation of intermoult puffs is quite complex and, so far, largely not understood. In order to further investigate this aspect, we have analysed the effects of mutations in a key regulator of the ecdysone response at the onset of metamorphosis, the Broad-Complex (BR-C) locus, on the expression of genes mapping at the 3C intermoult puff. On the basis of an accurate examination of 3C intermoult gene activity in single, carefully staged, third instar larvae of wild-type and BR-C mutant strains, we were able to subdivide these genes into two groups. Each group is characterised by a different temporal expression profile, so that at the beginning of the wandering stage the transcription of the first group declines as group II transcription is induced. Interestingly, the BR-C locus appears to play a regulatory role in establishing this transcriptional switch. By using mutants of each of the three lethal complementation groups, we precisely defined the role of BR-C functions in this developmental transition and we show that this locus also plays an essential role in the early pre-metamorphic hormonal response.

Real-time PCR quantification of human DKC1 expression in colorectal cancer

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Energy independent uptake and release of polystyrene nanoparticles in primary mammalian cell cultures

Nanoparticle (NPs) delivery systems in vivo promises to overcome many obstacles associated with the administration of drugs, vaccines, plasmid DNA and RNA materials, making the study of their cellular uptake a central issue in nanomedicine. The uptake of NPs may be influenced by the cell culture stage and the NPs physical-chemical properties. So far, controversial data on NPs uptake have been derived owing to the heterogeneity of NPs and the general use of immortalized cancer cell lines that often behave differently from each other and from primary mammalian cell cultures. Main aims of the present study were to investigate the uptake, endocytosis pathways, intracellular fate and release of well standardized model particles, i.e. fluorescent 44 nm polystyrene NPs (PS-NPs), on two primary mammalian cell cultures, i.e. bovine oviductal epithelial cells (BOEC) and human colon fibroblasts (HCF) by confocal microscopy and spectrofluorimetric analysis. Different drugs and conditions that inhibit specific internalization routes were used to understand the mechanisms that mediate PS-NP uptake. Our data showed that PS-NPs are rapidly internalized by both cell types 1) with similar saturation kinetics; 2) through ATP-independent processes, and 3) quickly released in the culture medium. Our results suggest that PS-NPs are able to rapidly cross the cell membrane through passive translocation during both uptake and release, and emphasize the need to carefully design NPs for drug delivery, to ensure their selective uptake and to optimize their retainment in the targeted cells.

A new human dyskerin isoform with cytoplasmic localization

BACKGROUND The human DKC1 gene is causative of X-linked dyskeratosis congenita (X-DC), a syndrome characterized by mucocutaneous features, bone marrow failure, tumor susceptibility, perturbation of stem cell function, and premature aging. DKC1 is thought to produce a single protein, named dyskerin, which shows strict nucleolar localization and participates in at least two distinct nuclear functional complexes: the H/ACA small nucleolar ribonucleoproteic complex involved in RNA pseudouridylation and the active telomerase complex. METHODS By bioinformatics and molecular analyses we identified a DKC1 splice variant able to encode a truncated form of dyskerin, confirmed its active expression in diverse human tissues by RT-PCR, and showed by immunoblotting and immunocytochemistry experiments that it actually encodes a novel protein. Stably transfected clones over-expressing the new isoform were analyzed for growth, morphology and adhesion properties. RESULTS Our results show that DKC1 encodes a new alternatively spliced mRNA able to direct the synthesis of a variant dyskerin with unexpected cytoplasmic localization. Intriguingly, when over-expressed in HeLa cells, the new isoform promotes cell to cell and cell to substratum adhesion, increases the cell proliferation rate and leads to cytokeratin hyper-expression. CONCLUSIONS AND GENERAL SIGNIFICANCE Our results highlight a novel degree of complexity and regulation of the human DKC1 gene and reveal that it can play a further, unpredicted role in cell adhesion. The identification of a dyskerin cytoplasmic variant reinforces the view that other mechanisms, in addition to telomere instability, can significantly contribute to the pathogenesis of the X-DC, and suggests that DKC1 nucleolar and cytoplasmic functions might cumulatively account for the plethora of manifestations displayed by this syndrome.

Linking pseudouridine synthases to growth, development and cell competition

Eukaryotic pseudouridine synthases direct RNA pseudouridylation and bind H/ACA small nucleolar RNA (snoRNAs), which, in turn, may act as precursors of microRNA‐like molecules. In humans, loss of pseudouridine synthase activity causes dyskeratosis congenita (DC), a complex systemic disorder characterized by cancer susceptibility, failures in ribosome biogenesis and telomere stability, and defects in stem cell formation. Considering the significant interest in deciphering the various molecular consequences of pseudouridine synthase failure, we performed a loss of function analysis of minifly (mfl), the pseudouridine synthase gene of Drosophila, in the wing disc, an advantageous model system for studies of cell growth and differentiation. In this organ, depletion of the mfl‐encoded pseudouridine synthase causes a severe reduction in size by decreasing both the number and the size of wing cells. Reduction of cell number was mainly attributable to cell death rather than reduced proliferation, establishing that apoptosis plays a key role in the development of the loss of function mutant phenotype. Depletion of Mfl also causes a proliferative disadvantage in mosaic tissues that leads to the elimination of mutant cells by cell competition. Intriguingly, mfl silencing also triggered unexpected effects on wing patterning and cell differentiation, including deviations from normal lineage boundaries, mingling of cells of different compartments, and defects in the formation of the wing margin that closely mimic the phenotype of reduced Notch activity. These results suggest that a component of the pseudouridine synthase loss of function phenotype is caused by defects in Notch signalling.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Loss of Drosophila pseudouridine synthase triggers apoptosis-induced proliferation and promotes cell-nonautonomous EMT.

Many developing tissues display regenerative capability that allows them to compensate cell loss and preserve tissue homeostasis. Because of their remarkable regenerative capability, Drosophila wing discs are extensively used for the study of regenerative phenomena. We thus used the developing wing to investigate the role played in tissue homeostasis by the evolutionarily conserved eukaryotic H/ACA small nucleolar ribonucleoprotein pseudouridine synthase. Here we show that localized depletion of this enzyme can act as an endogenous stimulus capable of triggering apoptosis-induced proliferation, and that context-dependent effects are elicited in different sub-populations of the silenced cells. In fact, some cells undergo apoptosis, whereas those surrounding the apoptotic foci, although identically depleted, overproliferate. This overproliferation correlates with ectopic induction of the Wg and JAK-STAT (Janus kinase-signal transducer and activator of transcription) mitogenic pathways. Expression of a p35 transgene, which blocks the complete execution of the death program and generates the so-called ‘undead cells’, amplifies the proliferative response. Pseudouridine synthase depletion also causes loss of apicobasal polarity, disruption of adherens cell junctions and ectopic induction of JNK (c-Jun N-terminal kinase) and Mmp1 (matrix metalloproteinase-1) activity, leading to a significant epithelial reorganization. Unexpectedly, cell-nonautonomous effects, such as epithelial mesenchymal transition in the contiguous unsilenced squamous epithelium, are also promoted. Collectively, these data point out that cell–cell communication and long-range signaling can take a relevant role in the response to pseudouridine synthase decline. Considering that all the affected pathways are highly conserved throughout evolution, it is plausible that the response to pseudouridine synthase depletion has been widely preserved. On this account, our results can add new light on the still unexplained tumor predisposition that characterizes X-linked dyskeratosis, the human disease caused by reduced pseudouridine synthase activity.

A novel Drosophila antisense scaRNA with a predicted guide function

A significant portion of eukaryotic small ncRNA transcriptome is composed by small nucleolar RNAs. From archaeal to mammalian cells, these molecules act as guides in the site-specific pseudouridylation or methylation of target RNAs. We used a bioinformatics search program to detect Drosophila putative orthologues of U79, one out of ten snoRNAs produced by GAS5, a human ncRNA involved in apoptosis, susceptibility to cancer and autoimmune diseases. This search led to the definition of a list of U79-related fly snoRNAs whose genomic organization, evolution and expression strategy are discussed here. We report that an intriguing novel specimen, named Dm46E3, is transcribed as a longer, unspliced precursor from the reverse strand of eiger, a fly regulatory gene that plays a key role in cell differentiation, apoptosis and immune response. Expression of Dm46E3 was found significantly up-regulated in a mutant strain in which eiger transcription is greatly reduced, suggesting that these two sense-antisense genes may be mutually regulated. Relevant to its function, Dm46E3 concentrated specifically in the Cajal bodies, followed a dynamic spatial expression profile during embryogenesis and displayed a degenerate antisense element that enables it to target U1b, a developmentally regulated isoform of the U1 spliceosomal snRNA that is particularly abundant in embryos.