Drosophila dyskerin is required for somatic stem cell homeostasis

Drosophila represents an excellent model to dissect the roles played by the evolutionary conserved family of eukaryotic dyskerins. These multifunctional proteins are involved in the formation of H/ ACA snoRNP and telomerase complexes, both involved in essential cellular tasks. Since fly telomere integrity is guaranteed by a different mechanism, we used this organism to investigate the specific role played by dyskerin in somatic stem cell maintenance. To this aim, we focussed on Drosophila midgut, a hierarchically organized and well characterized model for stemness analysis. Surprisingly, the ubiquitous loss of the protein uniquely affects the formation of the larval stem cell niches, without altering other midgut cell types. The number of adult midgut precursor stem cells is dramatically reduced, and this effect is not caused by premature differentiation and is cell-autonomous. Moreover, a few dispersed precursors found in the depleted midguts can maintain stem identity and the ability to divide asymmetrically, nor show cell-growth defects or undergo apoptosis. Instead, their loss is mainly specifically dependent on defective amplification. These studies establish a strict link between dyskerin and somatic stem cell maintenance in a telomerase-lacking organism, indicating that loss of stemness can be regarded as a conserved, telomerase-independent effect of dyskerin dysfunction

Dyskerin Downregulation Can Induce ER Stress and Promote Autophagy via AKT-mTOR Signaling Deregulation

: Dyskerin is an evolutionarily conserved nucleolar protein implicated in a wide range of fundamental biological roles, including telomere maintenance and ribosome biogenesis. Germline mutations of DKC1, the human gene encoding dyskerin, cause the hereditary disorders known as X-linked dyskeratosis congenita (X-DC). Moreover, dyskerin is upregulated in several cancers. Due to the pleiotropic functions of dyskerin, the X-DC clinical features overlap with those of both telomeropathies and ribosomopathies. In this paper, we evaluate the telomerase-independent effects of dyskerin depletion on cellular physiology by using inducible DCK1 knockdown. This system allows the downregulation of DKC1 expression within a short timeframe. We report that, in these cellular systems, dyskerin depletion induces the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum, which in turn induces the activation of the PERK branch of the unfolded protein response. We also demonstrate that the PERK-eIF2a-ATF4-CHOP signaling pathway, activated by dyskerin downregulation, triggers a functional autophagic flux through the inhibition of the PI3K/AKT/mTOR pathway. By revealing a novel unpredicted connection between the loss of dyskerin, autophagy and UPR, our results establish a firm link between the lowering of dyskerin levels and the activation of the ER stress response, that plays a key role in the pathogenesis of several diseases

A functional connection between dyskerin and energy metabolism

The human DKC1 gene encodes dyskerin, an evolutionarily conserved nuclear protein whose overexpression represents a common trait of many types of aggressive sporadic cancers. As a crucial component of the nuclear H/ACA snoRNP complexes, dyskerin is involved in a variety of essential processes, including telomere maintenance, splicing efficiency, ribosome biogenesis, snoRNAs stabilization and stress response. Although multiple minor dyskerin splicing isoforms have been identified, their functions remain to be defined. Considering that low-abundance splice variants could contribute to the wide functional repertoire attributed to dyskerin, possibly having more specialized tasks or playing significant roles in changing cell status, we investigated in more detail the biological roles of a truncated dyskerin isoform that lacks the C-terminal nuclear localization signal and shows a prevalent cytoplasmic localization. Here we show that this dyskerin variant can boost energy metabolism and improve respiration, ultimately conferring a ROS adaptive response and a growth advantage to cells. These results reveal an unexpected involvement of DKC1 in energy metabolism, highlighting a previously underscored role in the regulation of metabolic cell homeostasis

The Drosophila H/ACA snoRNP pseudouridine synthase and its interplay with key developmental pathways

Loss of function of the DKC1 human gene causes the X-linked Dyskeratosis Congenital (X-DC) disease, whose main symptoms are abnormal skin pigmentation, nail dystrophy, mucosal leukoplakia, bone marrow failure and increased tumour susceptibility. DKC1 encodes a nucleolar protein, named dyskerin, whose sequence is characterized by a high degree of phylogenetic conservation. Eukaryal dyskerins represent one of the four proteic core components of the H/ACA small nucleolar RNA-associated ribonucleoprotein (snoRNP) complexes that are involved in rRNA processing, pseusourydilation of cellular RNAs, modulation of the efficiency of IRES-dependent translation, and stabilization of H/ACA snoRNAs. Besides participating in the formation of H/ACA snoRNPs, mammalian dyskerin also associates with telomeric RNA, which contains an H/ACA domain, being one of the essential components of the telomerase active complex. One of the main challenges posed by X-DC pathogenesis is distinguishing between the effects caused by telomere shortening from those caused by altered snoRNPs functioning. Given that Drosophila lacks telomerase, and Drosophila dyskerin, encoded by the Nop60b/minifly (mfl) gene, is highly related to its human counterpart, this organism can serve as an useful model to investigate the telomerase-independent effects caused by depletion of snoRNP pseudouridine synthases. In this thesis I evaluated the effects of in vivo localized mfl gene silencing on the development of the wing imaginal disc, which represents an excellent model to study the morphogenetic regulation of organ growth and patterning. I found that mfl silencing triggers a process of “apoptosis-induced proliferation” that is typical of regenerative phenomena. This process correlates with epithelial reorganization, that is marked by cytoskeletal remodeling, activation of the JNK pathway activity and transition of patches of cells from the epithelial to the mesenchimal state. Moreover, I observed that mfl silencing causes dysregulation of Notch signaling at the D/V boundary of the wing disc; this dysregulation cannot be attributed to apoptosis or to defective IRES- dependent translation of the Notch antagonist Hairless protein. Altogheter, the results obtained reveal for the first time a close link that connects eukaryotic dyskerins with Notch signaling and JNK pathway. On the basis of their evolutionary conservation, I speculate that these events could be r!esponsible for at least some of the symptoms shown by X-DC patients

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

Human dyskerin: beyond telomeres

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 IRESdependent 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