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.

Artificial selection began to override natural selection in domesticated wild boar and other species about 10,000 years ago. The intentional selection of a desired phenotypic trait is a complex process, and comes along with unexpected or even unwanted changes in other traits, because of epistatic gene effects, and ontogenetic con- straints. The loss of brain mass in domestic ungulates is related to selection for reduced reaction to external stimuli. Evolutionary losses in body structures and genes were once considered mostly irreversible, in keeping with Dollo’s law. Here we studied the biochemical and the histological functioning of the free-living pigs (FLPs) olfactory system, to see if and to what extent does FLPs regain a full sense of smell, as compared to the domestic pigs and wild boar Sus scrofa. In our samples both wild boars and FLPs have significantly larger brain per unit mass than domestic pigs, and FLPs’ brains are not significantly smaller than wild boar’s. Similarly, both wild boars and FLPs have signifi- cantly higher cell density than domestic pigs in the olfac- tory mucosa. Yet, at the functional level, olfactory marker protein and neuropeptide Y, both of which are important to the correct functioning of the sense of smell, are fully expressed only in wild boar. These results suggest that FLPs reacquired structural, but not the biochemical capa- bility in their olfactory system

SUMMARY Glutamate receptor auxiliary proteins control receptor distribution and function, ultimately controlling synapse assembly, maturation, and plasticity. At the Drosophila neuromuscular junction (NMJ), a synapse with both pre- and postsynaptic kainate-type glutamate receptors (KARs), we show that the auxiliary protein Neto evolved functionally distinct isoforms to modulate synapse development and homeostasis. Using genetics, cell biology, and electrophysiology, we demonstrate that Neto-α functions on both sides of the NMJ. In muscle, Neto-α limits the size of the postsynaptic receptor field. In motor neurons (MNs), Neto-α controls neurotransmitter release in a KAR-dependent manner. In addition, Neto-α is both required and sufficient for the presynaptic increase in neurotransmitter release in response to reduced postsynaptic sensitivity. This KAR-independent function of Neto-α is involved in activity-induced cytomatrix remodeling. We propose that Drosophila ensures NMJ functionality by acquiring two Neto isoforms with differential expression patterns and activities.

Dyskerin is an essential, conserved, multifunctional protein found in the nucleolus, whose loss of function causes the rare genetic diseases X‐linked dyskeratosis congenita and Hoyeraal‐Hreidarsson syndrome. To further investigate the wide range of dyskerin's biological roles, we set up stable cell lines able to trigger inducible protein knockdown and allow a detailed analysis of the cascade of events occurring within a short time frame. We report that dyskerin depletion quickly induces cytoskeleton remodeling and significant alterations in endocytic Ras‐related protein Rab‐5A/Rab11 trafficking. These effects arise in different cell lines well before the onset of telomere shortening, which is widely considered the main cause of dyskerin‐related diseases. Given that vesicular trafficking affects many homeostatic and differentiative processes, these findings add novel insights into the molecular mechanisms underlining the pleiotropic manifestation of the dyskerin loss‐of‐function phenotype.

Single‐cell RNA sequencing provides a new approach to an old problem: how to study cellular diversity in complex biological systems. This powerful tool has been instrumental in profiling different cell types and investigating, at the single‐cell level, cell states, functions, and responses. However, mining these data requires new analytical and statistical methods for high‐dimensional analyses that must be customized and adapted to specific goals. Here we present a custom multistage analysis pipeline which integrates modules contained in different R packages to ensure flexible, high‐quality RNA‐seq data analysis. We describe this workflow step by step, providing the codes, explaining the rationale for each function, and discussing the results and the limitations. We apply this pipeline to analyze different datasets of Drosophila larval ventral cords, identifying and describing rare cell types, such as astrocytes and neuroendocrine cells. This multistage analysis pipeline can be easily implemented by both novice and experienced scientists interested in neuronal and/or cellular diversity beyond the Drosophila model system. © 2021 US Government.

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.

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.

Drosophila provides a powerful genetic system and an excellent model to study the development and function of the nervous system. The fly's small brain and complex behavior has been instrumental in mapping neuronal circuits and elucidating the neural basis of behavior. The fast pace of fly development and the wealth of genetic tools has enabled systematic studies on cell differentiation and fate specification, and has uncovered strategies for axon guidance and targeting. The accessibility of neuronal structures and the ability to edit and manipulate gene expression in selective cells and/or synaptic compartments has revealed mechanisms for synapse assembly and neuronal connectivity. Recent advances in single‐cell RNA sequencing (scRNA‐seq) have further enhanced our appreciation and understanding of neuronal diversity in a fly brain. However, due to the small size of the fly brain and its constituent cells, scRNA‐seq methodologies require a few adaptations. Here, we describe a set of protocols optimized for scRNA‐seq analysis of the Drosophila larval ventral nerve cord, starting from tissue dissection and cell dissociation to cDNA library preparation, sequencing, and data analysis. We apply this workflow to three separate samples and detail the technical challenges associated with successful application of scRNA‐seq to studies on neuronal diversity. An accompanying article (Vicidomini, Nguyen, Choudhury, Brody, & Serpe, 2021) presents a custom multistage analysis pipeline that integrates modules contained in different R packages to ensure high‐flexibility, high‐quality RNA‐seq data analysis. These protocols are developed for Drosophila larval ventral nerve cord, but could easily be adapted to other tissues and model organisms. © 2021 U.S. Government. Basic Protocol 1: Dissection of larval ventral nerve cords and preparation of single‐cell suspensions Basic Protocol 2: Preparation and sequencing of single‐cell transcriptome libraries Basic Protocol 3: Alignment of raw sequencing data to indexed genome and generation of count matrices.