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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., Graphical abstract fx1, Highlights • Human dyskerin is an evolutionary conserved component of nuclear H/ACA snoRNPs. • The functional role of a truncated dyskerin isoform (Iso3) is analyzed. • Iso3 overexpression boosts energy metabolism and induces a ROS adaptive response. • Iso3 connects dyskerin with mitochondrial functionality and redox homeostasis.

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.

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.

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.

Identification of alternatively spliced transcripts produced by a gene is a crucial step in deciphering the bulk of its biological roles and the overall processes that regulate its activity. By using a combination of bioinformatic and molecular approaches we identified, cloned, and characterized 3 novel alternative splice isoforms derived from human dyskeratosis congenita 1 (hDKC1), an essential human gene causative of the X-linked dyskeratosis congenita disease and involved in multiple functions related to cell growth, proliferation, and telomere maintenance. Expression of the new isoforms, all characterized by intron retention, was confirmed by RT-PCR in a panel of diverse cell lines and normal human tissues, and despite the presence of premature termination codons, was not down-regulated by the mechanism of nonsense-mediated decay. Accumulation of these transcripts fluctuated distinctly in the diverse tissues and during in vitro differentiation of Caco2 cells, suggesting that their ratio may contribute to the gene functional diversity across different cell types. Intriguingly, the structure of one isoform leads to exonize an intronically encoded small nucleolar RNA (snoRNA), highlighting an additional layer of complexity that can contribute to overall gene regulation.

Mutations in the highly conserved human DKC1 gene cause the rare genetic disease X-linked recessive dyskeratosis congenita (X-DC). X-DC patients and DKC1 hypomorphic mutant mice show an increased susceptibility to cancer, so that it has been suggested that DKC1 can act as potent tumor suppressor. The nucleolar protein encoded by DKC1, named dyskerin, is one of the core components of the nucleolar ribonucleoprotein particles which include the small nucleolar RNAs belonging to the H/ACA family. Within H/ACA snoRNPs, dyskerin is known to play a crucial role in rRNA processing and pseudouridylation, being able to act as pseudouridine synthase. In addition, dyskerin has been recently identified as an essential component of the catalytically active human telomerase complex, together with the human telomerase reverse trascriptase (TERT) and the RNA component of telomerase. Since TERT has been indicated as a potential biomarker for colorectal cancer, we wished to evaluate whether dyskerin and TERT mRNA levels were similarly variable in colorectal cancer. In this study, matched samples of tumors and adjacent non-tumorous mucosa from 8 colorectal carcinomas were collected, and Real-time polymerase chain reaction was used to quantitate DKC1 and TERT expression levels. We found that in the colorectal adenocarcinomas DKC1 mRNA level was significantly higher than in the corresponding non-tumor mucosa (p=0.0004, one-sided t test). Instead, TERT exhibited a variable mRNA level, although in most cases it was up-regulated respect to the matched controls (p=0.066, one-sided t test). These data support the view that DKC1 expression may be considered a less variable and more sensible marker than TERT for proliferative colon disorders. Although analysis of a more exhaustive series of tumors is needed, we suggest that the evaluation of DKC1 expression can provide a useful clue in the diagnosis and prognosis of colorectal cancer.