A functional connection between dyskerin and energy metabolism

Abstract

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.

Keywords: DKC1; Energy metabolism; Mitochondria; PRDX-2; ROS signaling.

Graphical abstract

Fig. 1

Overexpression of dyskerin Isoform3 boosts respiratory rate and mitochondrial membrane potential. (A) Schematic organization of full-length dyskerin (Isoform1) and dyskerin Isoform 3, lacking the C-terminal NLS. Colored boxes indicate structural domains: DKCLD, associated N-terminal domain of dyskerin-like proteins of unknown function; TRUB_N, pseudouridine synthase catalytic domain; PUA, pseudouridine synthase and archaeosine transglycosylase RNA binding domain; orange boxes, lysine-arginine rich NLS sequences. (B) 3XF-Iso3 and control cells (2.5 * 10³–1 * 10⁴) were seeded in triplicate, incubated overnight to allow attachment and the following day subjected to MTT assay to measure cell proliferation and metabolic activities. The amount of precipitated formazan was quantified by absorbance and expressed as optical density. (C) Oxygen consumption rate of 3XF-Iso3 and control cells was measured by Clark's electrode. Note that respiration, expressed as nmol O₂/min * 10⁶ cells, was nearly doubled in 3XF-Iso3 cells. (D) Mitochondrial /nuclear DNA content was quantified by qPCR; the mitochondrial/nuclear DNA ratio is reported. In this experiment, the 16S rRNA coding region was amplified and normalized with respect to the TSH receptor (TSHR) single-copy nuclear gene. (E) On the left: LUT quantitative analysis of confocal images of 3XF-Iso3 and 3XF-Mock viable cells, captured under the same conditions, upon staining with MitoTracker Green (in gray), which is insensitive to mitochondrial ΔΨ and allows a direct visualization of the mitochondrial network. Sum of total confocal planes is shown; magnification: 40×; scale bars: 10 µm. The white-dashed squares are enlarged in the insets. On the right: to estimate the mitochondrial mass, the intensity of the MitoTracker Green fluorescent signal was calculated from the same confocal images; values obtained from the sum of total planes were normalized in respect to cell areas and expressed as Intensity/cell area ratio by ImageJ tools. Data derived from the analysis of n = 90 cells. (F) qRT-PCR analysis of PGC-1α and PPRC1 expression in 3XF-Iso3 and 3XF-Mock quiescent cells; GSS and HPRT1 were used as normalizing reference genes. (G) Histogram representative of PGC-1α expression in 3XF-Iso3 and 3XF-Mock quiescent cells, as derived from western blotting densitometric quantification normalized with respect to β-tubulin (original data in Supplemental Fig. 1A). (H) Mitochondrial ΔΨ determined by flow cytometry analysis of TMRE stained cells. The TMRE dye permeates and is sequestered in active mitochondria, so that the amount of sequestered dye is directly dependent on mitochondrial ΔΨ. On the left, data derived from three different experiments; on the right, one representative experiment. Gate 1 represents the population identified as “cells”; gate 2 the “stained” population. In the right histogram, fluorescence intensity is plotted vs. cells count. All experiments were performed in triplicate; in B–D, F–H data are expressed as the mean ± SD.

Fig. 2

Redox state and ROS induction in Isoform 3 over-expressing cells. (A) Cells were stained with propidium iodide and analyzed by flow cytometry in the absence (−) or following overnight treatment with 0.25 µM rotenone (+). Percentages refer to the total number of examined cells, while plots compare an equal number (10.000) of 3XF-Iso3 and control cells. Note that the fraction of hypodiploid cells does not increase in untreated 3XF-Iso3 cells, indicating that Iso3 overexpression per se does not affect the healthy state of cell population; in contrast, this fraction significantly increases in the 3XF-Iso3 cell population upon rotenone treatment. (B) Cellular redox status analyzed by live multiconfocal microscopy following near-infra red excitation. Upper panel shows confocal micrographs of FAD (green) and NAD(P)H (red) autofluorescence signals; lower table reports statistical analysis of fluorescence signals from at least 50 analyzed ROI, selected on the basis of their high mitochondrial content; values are expressed as gray scale units. Scale bars: 10 µm. (C) 3XF-Iso3 cells are more sensitive to malonate treatment compared to controls. In both cases, the treatment induces a marked change in cells (on the left). (D–E) Total ROS and mitochondrial superoxide levels were measured upon H2DCFDA or MitoSOX Red staining and flow cytometry analysis. Right inset (red rectangle) in D highlights the increase of the superoxide high-producer cell fraction present in 3XF-Iso3 population, identified as “% cells gate 2” in the exp 1 reported in the enclosed table.

Fig. 3

Enhanced expression of mitochondrial proteins and detoxification enzymes contribute to the healthy state of Iso3 over-expressing cells. On the top are histograms representative of expression levels of ETC components and key mitochondrial proteins. Data derived from western blotting densitometric quantification, normalized with respect to β-tubulin (original data in Supplemental Fig. 1B); Note that NDUFS3 (Complex I), SDHb (complex II), CYSC (Complex III) levels all increased in 3XF-Iso3 cells; instead, expression of DRP1, which promotes mitochondrial fission, and of OPA1, a pro-fusion protein that produces long (L-OPA1) and short (S-OPA1) isoforms (bottom), remained substantially unvaried. Expression of TOM20, a component of the outer mitochondrial membrane, was significantly up-regulated in 3XF-Iso3 cells. (B) LUT quantitative analysis of confocal images, captured under the same conditions, depicting TOM20 expression in 3XF-Iso3 and 3XF-Mock cells. The anti-TOM20 antibody (green/gray) stains the mitochondrial network; Phalloidin (red) marks the actin cytoskeleton; DAPI (blue) counterstains the nuclei. Images are at 63× magnification; scale bars: 10 µm; the sum of total confocal planes is shown. On the right: intensity signal values obtained from the sum of total planes were normalized with respect to F-actin signal and expressed as TOM20 intensity/actin intensity ratio by ImageJ tools. Data derived from the analysis of n = 50 cells. (C) Histograms representative of expression levels of a panel of detoxifying enzymes. Data derived from western blotting densitometric quantification, normalized with respect to β-tubulin (original data in Supplemental Fig. 1C). (D) LUT quantitative analysis of confocal images, captured under the same conditions, of 3XF-Iso3 and 3XF-Mock cells stained with anti-PRDX-2 (gray); nuclei are counterstained by DAPI (blue). Sum of total planes is shown; magnification is at 63×; scale bars: 10 µm. On the right: intensity signal values obtained from the sum of total planes were normalized with respect to cell areas and expressed as Intensity/cell area ratio by ImageJ tools. Data derived from the analysis of n = 50 cells. (E) Confocal images of 3XF-Iso3 and 3XF-Mock cells upon staining with the anti-AIFM1 antibody (green); nuclei are counterstained by DAPI (blue). On the left, maximum projection of z-stack confocal planes; on the right, maximum projection of z-stack, flanked by orthogonal views. Scale bars: 10 µm. In A, C, blotting were performed in triplicate, and data expressed as the mean ± SD.