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Philip J. Mason - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked
2016Co-Authors: Dyskeratosis Congenita Patients, Monica Bessler, Jian-meng Fan, Marisa Apicella, Jason Mills, Dara A. Reeves, Deborah French, Gregory M. Podsakoff, Philip J. MasonAbstract:Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by the presence of short telomeres at presentation. Mutations in ten different genes, whose products are involved in the telomere maintenance pathway, have been shown to cause DC. The X-linked form is the most common form of the disease and is caused by mutations in the gene DKC1, encoding the protein Dyskerin. Dyskerin is required for the assembly and stability of telomerase and is also involved in ribosomal RNA (rRNA) processing where it converts specific uridines to pseudouridine. DC is thought to result from failure to maintain tissues, like blood, that are renewed by stem cell activity, but research into pathogenic mechanisms has been hampered by the difficulty of obtaining stem cells from patients. We reasoned that induced pluripotent stem (iPS) cells from X-linked DC patients may provide information about the mechanisms involved. Here we describe the production of iPS cells from DC patients with DKC1mutations Q31E, A353Vand ΔL37. In addition we constructed “corrected ” lines with a copy of the wild type Dyskerin cDNA expressed from the AAVS1 safe harbor locus. We show that in iPS cells with DKC1mutations telomere maintenanc
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Expression of WT Dyskerin can rescue the expression of LGR5, WLS and FRZB.
2015Co-Authors: Marisa Apicella, Monica Bessler, Jian-meng Fan, Jason Mills, Dara A. Reeves, Deborah French, Gregory M. Podsakoff, Philip J. MasonAbstract:A: Real-Time RT/PCR experiments showed that, in A353V iPS cells, the mRNA expression of LGR5, FRZB and WLS was significantly increased after expressing WT Dyskerin protein. The combined results of 3 independent experiments are shown, the error bars show standard deviation. * p
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Expression of WT Dyskerin can rescue dysfunctional telomerase in ΔL37 cells but not in A353V cells.
2015Co-Authors: Marisa Apicella, Monica Bessler, Jian-meng Fan, Jason Mills, Dara A. Reeves, Deborah French, Gregory M. Podsakoff, Philip J. MasonAbstract:A: Schematic representation of zinc-finger nuclease (ZNF)-mediated homologous recombination. The constitutively active AAVS1 “safe harbor” locus is shown on the top line and the targeting construct is shown below. The cDNA expression cassettes driving expression of Flag-tagged WT Dyskerin under the chicken actin promoter (CAGG) were inserted by zinc finger-mediated homologous recombination into intron 1 of AAVS1. HA, homologous arms left (L) and right (R); SA-2APuro-PA, puromycin drug resistance cassette. [30] B: Western blot showing expression of flag-tagged Dyskerin protein in corrected A353V and ΔL37 iPS cells by using anti-Flag antibody and anti-Dyskerin antibody. Open arrow: endogenous Dyskerin protein; filled arrow: Flag-tagged WT Dyskerin protein. C: Northern blot result of TERC RNA expression levels of DKC1 corrected iPS cells. Different corrected lines are shown as well as the uncorrected line carrying the DKC1 ΔL37 or the DKC1 A353V mutation. β-actin was used as loading control. The quantitive data, derived by densitometry, are shown. D: The TRAP assay was performed to measure the telomerase activity after expressing the WT DKC1 gene in A353V and ΔL37 iPS cells, uncorrected and corrected by ectopic expression of WT DKC1 (DKC1-C). The quantitive data, derived by densitometry, are shown E: Telomere lengths of DKC1 corrected iPS cells were measured by using in-gel hybridization with a telomere probe (TTAGGG)3. DKC1-C indicates corrected iPS cells expressing WT DKC1 gene.
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Slow growth and unstable ribosomal RNA lacking pseudouridine in mouse embryonic fibroblast cells expressing catalytically inactive Dyskerin
FEBS letters, 2013Co-Authors: Jian-meng Fan, Monica Bessler, Philip J. MasonAbstract:Pseudouridine is the most abundant modified nucleotide in ribosomal RNA throughout eukaryotes and archaea but its role is not known. Here we produced mouse embryonic fibroblast cells expressing only catalytically inactive Dyskerin, the pseudouridine synthase that converts uridine to pseudouridine in ribosomal RNA. The mutant Dyskerin protein, D125A, was extremely unstable but cells were able to divide and grow very slowly. Abnormalities in ribosome RNA synthesis were apparent but mature cytoplasmic RNAs lacking pseudouridine were produced and were very unstable. We conclude that pseudouridine is required to stabilize the secondary structure of ribosomal RNA that is essential for its function.
Leandro Sastre - One of the best experts on this subject based on the ideXlab platform.
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Dyskerin mutations present in dyskeratosis congenita patients increase oxidative stress and dna damage signalling in dictyostelium discoideum
Cells, 2019Co-Authors: Javier Rodriguezcenteno, Rosario Perona, Leandro SastreAbstract:Dyskerin is a protein involved in the formation of small nucleolar and small Cajal body ribonucleoproteins. These complexes participate in RNA pseudouridylation and are also components of the telomerase complex required for telomere elongation. Dyskerin mutations cause a rare disease, X-linked dyskeratosis congenita, with no curative treatment. The social amoeba Dictyostelium discoideum contains a gene coding for a Dyskerin homologous protein. In this article D. discoideum mutant strains that have mutations corresponding to mutations found in dyskeratosis congenita patients are described. The phenotype of the mutant strains has been studied and no alterations were observed in pseudouridylation activity and telomere structure. Mutant strains showed increased proliferation on liquid culture but reduced growth feeding on bacteria. The results obtained indicated the existence of increased DNA damage response and reactive oxygen species, as also reported in human Dyskeratosis congenita cells and some other disease models. These data, together with the haploid character of D. discoideum vegetative cells, that resemble the genomic structure of the human Dyskerin gene, located in the X chromosome, support the conclusion that D. discoideum can be a good model system for the study of this disease.
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gse4 a small Dyskerin and gse24 2 related peptide induces telomerase activity cell proliferation and reduces dna damage oxidative stress and cell senescence in Dyskerin mutant cells
PLOS ONE, 2015Co-Authors: Laura Iarriccio, Cristina Manguangarcia, Laura Pintadoberninches, Jose M Mancheno, Antonio J Molina, Rosario Perona, Leandro SastreAbstract:Dyskeratosis congenita is an inherited disease caused by mutations in genes coding for telomeric components. It was previously reported that expression of a Dyskerin-derived peptide, GSE24.2, increases telomerase activity, regulates gene expression and decreases DNA damage and oxidative stress in dyskeratosis congenita patient cells. The biological activity of short peptides derived from GSE24.2 was tested and one of them, GSE4, that probed to be active, was further characterized in this article. Expression of this eleven amino acids long peptide increased telomerase activity and reduced DNA damage, oxidative stress and cell senescence in Dyskerin-mutated cells. GSE4 expression also activated c-myc and TERT promoters and increase of c-myc, TERT and TERC expression. The level of biological activity of GSE4 was similar to that obtained by GSE24.2 expression. Incorporation of a Dyskerin nuclear localization signal to GSE24.2 did not change its activity on promoter regulation and DNA damage protection. However, incorporation of a signal that increases the rate of nucleolar localization impaired GSE24.2 activity. Incorporation of the Dyskerin nuclear localization signal to GSE4 did not alter its biological activity. Mutation of the Aspartic Acid residue that is conserved in the pseudouridine synthase domain present in GSE4 did not impair its activity, except for the repression of c-myc promoter activity and the decrease of c-myc, TERT and TERC gene expression in Dyskerin-mutated cells. These results indicated that GSE4 could be of great therapeutic interest for treatment of dyskeratosis congenita patients.
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expression of the genetic suppressor element 24 2 gse24 2 decreases dna damage and oxidative stress in x linked dyskeratosis congenita cells
PLOS ONE, 2014Co-Authors: Cristina Manguangarcia, Laura Pintadoberninches, Leandro Sastre, Rosario Machadopinilla, Jaime Carrillo, Carme Perezquilis, Isabel EsmorisAbstract:The predominant X-linked form of Dyskeratosis congenita results from mutations in DKC1, which encodes Dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of Dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. Here we have found that an increased basal and induced DNA damage response occurred in X-DC cells in comparison with normal cells. DNA damage that is also localized in telomeres results in increased heterochromatin formation and senescence. Expression of a cDNA coding for GSE24.2 rescues both global and telomeric DNA damage. Furthermore, transfection of bacterial purified or a chemically synthesized GSE24.2 peptide is able to rescue basal DNA damage in X-DC cells. We have also observed an increase in oxidative stress in X-DC cells and expression of GSE24.2 was able to diminish it. Altogether our data indicated that supplying GSE24.2, either from a cDNA vector or as a peptide reduces the pathogenic effects of Dkc1 mutations and suggests a novel therapeutic approach.
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congenita and in telomerase deficient human cells a Dyskerin motif reactivates telomerase activity in x linked dyskeratosis
2013Co-Authors: Rosario Machadopinilla, Isabel Sanchezperez, Jose Ramon Murguia, Leandro SastreAbstract:Abstract Dyskerin gene is mutated in X-linked dyskeratosis congenita patients (X-DC) which results in greatly reduced levels of telomerase activity. A genetic suppressor element (GSE) termed GSE24-2 has been isolated in a screening for cisplatin resistance. GSE24-2 expressing cells presented impaired telomerase inhibition following in vitro exposure to chemotherapies, such as cisplatin, or telomerase inhibitors. The promoter of the telomerase component hTERT, was constitutively activated in GSE24-2 cells in a c-myc expression-dependent manner. Deletion analyses and mutagenesis of the human c-myc promoter demonstrated that the target sequence for activation was the NHEIII site located upstream to the P1 region of the promoter. Further, expression of GSE24-2 in cell lines derived from X-DC patients and in VA13 cells induced increased hTERT RNA and hTR levels and recovery of telomerase activity. Finally, expression of GSE24-2 was able to rescue X-DC fibroblasts from premature senescence. These data demonstrate that this domain of Dyskerin plays an important role in telomerase maintenance following cell insults such as cisplatin treatment, and in telomerase-defective cells in patients with X-DC. The expression of this Dyskerin fragment has a dominant function in X-DC cells and could provide the basis for a therapeutic approach to this disease. From bloodjournal.hematologylibrary.org by guest on June 7, 2013. For personal use only.
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defects in mtr stability and telomerase activity produced by the dkc1 a353v mutation in dyskeratosis congenita are rescued by a peptide from the Dyskerin trub domain
Clinical & Translational Oncology, 2012Co-Authors: Rosario Machadopinilla, Philip J. Mason, Cristina Manguangarcia, Leandro Sastre, Jaime Carrillo, Alexander J Mentzer, Rosario PeronaAbstract:The predominant X-linked form of dyskeratosis congenita results from mutations in Dyskerin, a protein required for ribosomal RNA modification that is also a component of the telomerase complex. We have previously found that expression of an internal fragment of Dyskerin (GSE24.2) rescues telomerase activity in X-linked dyskeratosis congenita (X-DC) patient cells. Here, we have generated F9 mouse cell lines expressing the most frequent mutation found in X-DC patients, A353V and study the effect of expressing the GSE24.2 cDNA or GSE24.2 peptide on telomerase activity by TRAP assay, and mTERT and mTR expression by Q-PCR. Point mutation in GSE24.2 residues were generated by site-directed mutagenesis. Expression of GSE24.2 increases mTR and to a lesser extent mTERT RNA levels, and leads to recovery of telomerase activity. Point mutations in GSE24.2 residues known to be highly conserved and crucial for the pseudouridine-synthase activity of Dyskerin abolished the effect of the peptide. Recovery of telomerase activity and increase in mTERT levels were found when the GSE24.2 peptide purified from bacteria was introduced into the cells. Moreover, mTR stability was also rescued by transfection of the peptide GSE24.2. These data indicate that supplying GSE24.2, either from a cDNA vector, or as a peptide, can reduces the pathogenic effects of Dkc1 mutations and could form the basis of a novel therapeutic approach.
Lorenzo Montanaro - One of the best experts on this subject based on the ideXlab platform.
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cancer
2016Co-Authors: Marianna Penzo, Davide Trere, Vienna Ludovini, Annamaria Siggillino, Guido Bellezza, Lucio Crino, Lorenzo MontanaroAbstract:www.impactjournals.com/oncotarget / Oncotarget, Vol. 6, No. 25 Dyskerin and TERC expression may condition survival in lun
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Dyskerin and terc expression may condition survival in lung cancer patients
Oncotarget, 2015Co-Authors: Marianna Penzo, Davide Trere, Vienna Ludovini, Annamaria Siggillino, Jacopo Vannucci, Guido Bellezza, Lucio Crino, Lorenzo MontanaroAbstract:Dyskerin mediates both the modification of uridine on ribosomal and small nuclear RNAs and the stabilization of the telomerase RNA component (TERC). In human tumors Dyskerin expression was found to be associated with both rRNA modification and TERC levels. Moreover, Dyskerin overexpression has been linked to unfavorable prognosis in a variety of tumor types, however an explanation for the latter association is not available. To clarify this point, we analyzed the connection between Dyskerin expression, TERC levels and clinical outcome in two series of primary lung cancers, differing for the presence of TERC gene amplification, a genetic alteration inducing strong TERC overexpression. TERC levels were significantly higher in tumors bearing TERC gene amplification (P = 0.017). In addition, the well-established association between Dyskerin expression and TERC levels was observed only in the series without TERC gene amplification (P = 0.003), while it was not present in TERC amplified tumors (P = 0.929). Similarly, the association between Dyskerin expression and survival was found in cases not bearing TERC gene amplification (P = 0.009) and was not observed in TERC amplified tumors (P = 0.584). These results indicate that the influence of Dyskerin expression on tumor clinical outcome is linked to its role on the maintenance of high levels of TERC.
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human ribosomes from cells with reduced Dyskerin levels are intrinsically altered in translation
The FASEB Journal, 2015Co-Authors: Marianna Penzo, Laura Rocchi, Maurizio Brigotti, Sabine Brugiere, Domenica Carnicelli, Carmine Onofrillo, Yohann Coute, Lorenzo MontanaroAbstract:Dyskerin is a pseudouridine (ψ) synthase involved in fundamental cellular processes including uridine modification in rRNA and small nuclear RNA and telomere stabilization. Dyskerin functions are altered in X-linked dyskeratosis congenita (X-DC) and cancer. Dyskerin's role in rRNA pseudouridylation has been suggested to underlie the alterations in mRNA translation described in cells lacking Dyskerin function, although relevant direct evidences are currently lacking. Our purpose was to establish definitely whether defective Dyskerin function might determine an intrinsic ribosomal defect leading to an altered synthetic activity. Therefore, ribosomes from Dyskerin-depleted human cells were purified and 1) added to a controlled reticulocyte cell-free system devoid of ribosomes to study mRNA translation; 2) analyzed for protein contamination and composition by mass spectrometry, 3) analyzed for global pseudouridylation levels. Ribosomes purified from Dyskerin-depleted cells showed altered translational fidelit...
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Dyskerin expression in human fetal adult and neoplastic intrahepatic bile ducts correlations with cholangiocarcinoma aggressiveness
Histopathology, 2015Co-Authors: Francesco Vasuri, Laura Rocchi, Alessio Degiovanni, Francesca Giunchi, Giovanni Brandi, Davide Trere, Lorenzo Montanaro, Antonia DerricogrigioniAbstract:Aims To investigate the immunohistochemical expression of Dyskerin, a biomarker involved in ribosome production and telomere maintenance, in human fetal, adult and neoplastic bile ducts, and possible correlations with cholangiocarcinoma aggressiveness. Methods and results Sixty consecutive intrahepatic cholangiocarcinomas were collected and used for tissue microarray construction (total: 176 cores); clinical data and follow-up were also collected. Five fetal and 10 normal adult livers were included as controls. Automated immunohistochemistry for Dyskerin, p53, and Ki67, and nucleolar silver staining, were performed. In normal livers, Dyskerin expression was negative in smaller bile ducts (mean 44.8 μm) and positive in bile ducts of larger diameter (mean 116.1 μm; P < 0.001). Expression was positive in 56.7% of cholangiocarcinomas, and correlated with p53 mutation (P = 0.008) and a higher proliferative (Ki67) index (P = 0.003), which were included as markers of tumour aggressiveness. Finally, Dyskerin-positive cholangiocarcinomas showed a negative trend in disease-free survival (P = 0.078) on univariate analysis. Conclusions The non-neoplastic biliary tree seems to progressively lose Dyskerin expression from the major branches to the peripheral portal bile ducts. Similarly, intrahepatic cholangiocarcinomas showed two patterns of Dyskerin expression, and the Dyskerin-positive phenotype seemed to characterize more aggressive cholangiocarcinomas.
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inhibition of human Dyskerin as a new approach to target ribosome biogenesis
PLOS ONE, 2014Co-Authors: Laura Rocchi, Carmine Onofrillo, Armenio Jorge Moura Barbosa, Alberto Del Rio, Lorenzo MontanaroAbstract:The product of the DKC1 gene, Dyskerin, is required for both ribosome biogenesis and telomerase complex stabilization. Targeting these cellular processes has been explored for the development of drugs to selectively or preferentially kill cancer cells. Presently, intense research is conducted involving the identification of new biological targets whose modulation may simultaneously interfere with multiple cellular functions that are known to be hyper-activated by neoplastic transformations. Here, we report, for the first time, the computational identification of small molecules able to inhibit Dyskerin catalytic activity. Different in silico techniques were applied to select compounds and analyze the binding modes and the interaction patterns of ligands in the human Dyskerin catalytic site. We also describe a newly developed and optimized fast real-time PCR assay that was used to detect Dyskerin pseudouridylation activity in vitro. The identification of new Dyskerin inhibitors constitutes the first proof of principle that the pseudouridylation activity can be modulated by means of small molecule agents. Therefore, the presented results, obtained through the usage of computational tools and experimental validation, indicate an alternative therapeutic strategy to target ribosome biogenesis pathway.
Monica Bessler - One of the best experts on this subject based on the ideXlab platform.
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RESEARCH ARTICLE Impaired Telomere Maintenance and Decreased Canonical WNT Signaling but Normal Ribosome Biogenesis in Induced Pluripotent Stem Cells from X-Linked
2016Co-Authors: Dyskeratosis Congenita Patients, Monica Bessler, Jian-meng Fan, Marisa Apicella, Jason Mills, Dara A. Reeves, Deborah French, Gregory M. Podsakoff, Philip J. MasonAbstract:Dyskeratosis congenita (DC) is an inherited bone marrow failure syndrome characterized by the presence of short telomeres at presentation. Mutations in ten different genes, whose products are involved in the telomere maintenance pathway, have been shown to cause DC. The X-linked form is the most common form of the disease and is caused by mutations in the gene DKC1, encoding the protein Dyskerin. Dyskerin is required for the assembly and stability of telomerase and is also involved in ribosomal RNA (rRNA) processing where it converts specific uridines to pseudouridine. DC is thought to result from failure to maintain tissues, like blood, that are renewed by stem cell activity, but research into pathogenic mechanisms has been hampered by the difficulty of obtaining stem cells from patients. We reasoned that induced pluripotent stem (iPS) cells from X-linked DC patients may provide information about the mechanisms involved. Here we describe the production of iPS cells from DC patients with DKC1mutations Q31E, A353Vand ΔL37. In addition we constructed “corrected ” lines with a copy of the wild type Dyskerin cDNA expressed from the AAVS1 safe harbor locus. We show that in iPS cells with DKC1mutations telomere maintenanc
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Expression of WT Dyskerin can rescue dysfunctional telomerase in ΔL37 cells but not in A353V cells.
2015Co-Authors: Marisa Apicella, Monica Bessler, Jian-meng Fan, Jason Mills, Dara A. Reeves, Deborah French, Gregory M. Podsakoff, Philip J. MasonAbstract:A: Schematic representation of zinc-finger nuclease (ZNF)-mediated homologous recombination. The constitutively active AAVS1 “safe harbor” locus is shown on the top line and the targeting construct is shown below. The cDNA expression cassettes driving expression of Flag-tagged WT Dyskerin under the chicken actin promoter (CAGG) were inserted by zinc finger-mediated homologous recombination into intron 1 of AAVS1. HA, homologous arms left (L) and right (R); SA-2APuro-PA, puromycin drug resistance cassette. [30] B: Western blot showing expression of flag-tagged Dyskerin protein in corrected A353V and ΔL37 iPS cells by using anti-Flag antibody and anti-Dyskerin antibody. Open arrow: endogenous Dyskerin protein; filled arrow: Flag-tagged WT Dyskerin protein. C: Northern blot result of TERC RNA expression levels of DKC1 corrected iPS cells. Different corrected lines are shown as well as the uncorrected line carrying the DKC1 ΔL37 or the DKC1 A353V mutation. β-actin was used as loading control. The quantitive data, derived by densitometry, are shown. D: The TRAP assay was performed to measure the telomerase activity after expressing the WT DKC1 gene in A353V and ΔL37 iPS cells, uncorrected and corrected by ectopic expression of WT DKC1 (DKC1-C). The quantitive data, derived by densitometry, are shown E: Telomere lengths of DKC1 corrected iPS cells were measured by using in-gel hybridization with a telomere probe (TTAGGG)3. DKC1-C indicates corrected iPS cells expressing WT DKC1 gene.
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Expression of WT Dyskerin can rescue the expression of LGR5, WLS and FRZB.
2015Co-Authors: Marisa Apicella, Monica Bessler, Jian-meng Fan, Jason Mills, Dara A. Reeves, Deborah French, Gregory M. Podsakoff, Philip J. MasonAbstract:A: Real-Time RT/PCR experiments showed that, in A353V iPS cells, the mRNA expression of LGR5, FRZB and WLS was significantly increased after expressing WT Dyskerin protein. The combined results of 3 independent experiments are shown, the error bars show standard deviation. * p
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Slow growth and unstable ribosomal RNA lacking pseudouridine in mouse embryonic fibroblast cells expressing catalytically inactive Dyskerin
FEBS letters, 2013Co-Authors: Jian-meng Fan, Monica Bessler, Philip J. MasonAbstract:Pseudouridine is the most abundant modified nucleotide in ribosomal RNA throughout eukaryotes and archaea but its role is not known. Here we produced mouse embryonic fibroblast cells expressing only catalytically inactive Dyskerin, the pseudouridine synthase that converts uridine to pseudouridine in ribosomal RNA. The mutant Dyskerin protein, D125A, was extremely unstable but cells were able to divide and grow very slowly. Abnormalities in ribosome RNA synthesis were apparent but mature cytoplasmic RNAs lacking pseudouridine were produced and were very unstable. We conclude that pseudouridine is required to stabilize the secondary structure of ribosomal RNA that is essential for its function.
Inderjeet Dokal - One of the best experts on this subject based on the ideXlab platform.
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Single-Molecule Analysis of the Human Telomerase RNA·Dyskerin Interaction and the Effect of Dyskeratosis Congenita Mutations
2015Co-Authors: Beth Ashbridge, Inderjeet Dokal, Tom Vulliamy, Michael Kirwan, Angel Orte, Justin A Yeoman, David Klenerman, Shankar BalasubramanianAbstract:It has been proposed that human telomerase RNA (hTR) interacts with Dyskerin, prior to assembly of the telomerase holoenzyme. The direct interaction of Dyskerin and hTR has not been demonstrated and is an experimentally challenging research problem because of difficulties in expressing and purifying Dyskerin in quantities that are useful for biophysical analysis. By orthogonally labeling Dyskerin and hTR, we have been able to employ single-molecule two-color coincidence detection (TCCD) to observe directly the formation of a Dyskerin·hTR complex. By systematic deletion of hTR subdomains, we have gained insights into the RNA sites required for interaction with Dyskerin. We then investigated mutated forms of hTR and Dyskerin that are associated with dyskeratosis congenita (DC), on the basis of clinical genetics studies, for their effects on the Dyskerin·hTR interaction. Dyskerin mutations associated with X-linked DC resulted in significant impairment of the Dyskerin·hTR interaction, whereas mutations in hTR associated with autosomal dominant (AD) DC did not affect the interaction. We propose that disruption of the Dyskerin·hTR interaction may contribute to X-linked DC
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single molecule analysis of the human telomerase rna Dyskerin interaction and the effect of dyskeratosis congenita mutations
Biochemistry, 2009Co-Authors: Beth Ashbridge, Inderjeet Dokal, Tom Vulliamy, Michael Kirwan, Angel Orte, Justin A Yeoman, David Klenerman, Shankar BalasubramanianAbstract:It has been proposed that human telomerase RNA (hTR) interacts with Dyskerin, prior to assembly of the telomerase holoenzyme. The direct interaction of Dyskerin and hTR has not been demonstrated and is an experimentally challenging research problem because of difficulties in expressing and purifying Dyskerin in quantities that are useful for biophysical analysis. By orthogonally labeling Dyskerin and hTR, we have been able to employ single-molecule two-color coincidence detection (TCCD) to observe directly the formation of a Dyskerin·hTR complex. By systematic deletion of hTR subdomains, we have gained insights into the RNA sites required for interaction with Dyskerin. We then investigated mutated forms of hTR and Dyskerin that are associated with dyskeratosis congenita (DC), on the basis of clinical genetics studies, for their effects on the Dyskerin·hTR interaction. Dyskerin mutations associated with X-linked DC resulted in significant impairment of the Dyskerin·hTR interaction, whereas mutations in hTR...
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Mutations in the telomerase component NHP2 cause the premature ageing syndrome dyskeratosis congenita
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Tom Vulliamy, Anna Marrone, Amanda J. Walne, Richard Beswick, Michael Kirwan, Martin Digweed, Inderjeet DokalAbstract:Dyskeratosis congenita is a premature aging syndrome characterized by muco-cutaneous features and a range of other abnormalities, including early greying, dental loss, osteoporosis, and malignancy. Dyskeratosis congenita cells age prematurely and have very short telomeres. Patients have mutations in genes that encode components of the telomerase complex (Dyskerin, TERC, TERT, and NOP10), important in the maintenance of telomeres. Many dyskeratosis congenita patients remain uncharacterized. Here, we describe the analysis of two other proteins, NHP2 and GAR1, that together with Dyskerin and NOP10 are key components of telomerase and small nucleolar ribonucleoprotein (snoRNP) complexes. We have identified previously uncharacterized NHP2 mutations that can cause autosomal recessive dyskeratosis congenita but have not found any GAR1 mutations. Patients with NHP2 mutations, in common with patients bearing Dyskerin and NOP10 mutations had short telomeres and low TERC levels. SiRNA-mediated knockdown of NHP2 in human cells led to low TERC levels, but this reduction was not observed after GAR1 knockdown. These findings suggest that, in human cells, GAR1 has a different impact on the accumulation of TERC compared with Dyskerin, NOP10, and NHP2. Most of the mutations so far identified in patients with classical dyskeratosis congenita impact either directly or indirectly on the stability of RNAs. In keeping with this effect, patients with Dyskerin, NOP10, and now NHP2 mutations have all been shown to have low levels of telomerase RNA in their peripheral blood, providing direct evidence of their role in telomere maintenance in humans.
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dyskeratosis congenita advances in the understanding of the telomerase defect and the role of stem cell transplantation
Pediatric Transplantation, 2007Co-Authors: Josu De La Fuente, Inderjeet DokalAbstract:: DC is a multisystem bone marrow failure syndrome exhibiting marked clinical and genetic heterogeneity. X-linked, autosomal dominant and autosomal recessive subtypes are recognized. The gene mutated in X-linked DC (DKC1) encodes a highly conserved nucleolar protein called Dyskerin. Dyskerin associates with the H/ACA motif class of small nucleolar RNAs in small nucleolar ribonucleoprotein particles that are important in guiding the conversion of uracil to pseudouracil during the maturation of ribosomal RNA. Dyskerin also associates with the TERC, which is important in the maintenance of telomeres. Mutations in TERC have been identified in patients with autosomal dominant DC and in a subset of patients with aplastic anemia and myelodysplasia. Recently, heterozygous mutations in TERT have been found in some patients with autosomal dominant DC and aplastic anemia. Additionally, patients with the severe multisystem disorder, Hoyeraal–Hreidarsson syndrome, have been found to have DKC1 mutations. Collectively, these observations have demonstrated that classical DC, Hoyeraal–Hreidarsson syndrome and a subset of aplastic anemia are due to a primary defect in telomerase. The critical role of telomeres and telomerase in humans is seen in the multisystem abnormalities found in these patients, including the increased incidence of malignancy. As bone marrow failure is the principal cause of death, conventional allografts have been attempted with limited success due to the high rate of pulmonary and endothelial complications. However, outcomes have improved with the use of non-myeloablative protocols, although the follow up is too short to evaluate long term toxicity and the natural course of the disease and it may be that correction of the telomerase defect is essential for the treatment of these patients.
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Dyskeratosis congenita: molecular insights into telomerase function, ageing and cancer.
Expert reviews in molecular medicine, 2004Co-Authors: Anna Marrone, Inderjeet DokalAbstract:Dyskeratosis congenita (DC) is a severe, inherited, bone marrow failure syndrome, with associated cutaneous and noncutaneous abnormalities. DC patients also show signs of premature ageing and have an increased occurrence of cancer. DC can originate through: (1) mutations in DKC1, which result in X-linked recessive DC; (2) mutations in the RNA component of telomerase (TERC), which result in autosomal dominant DC (AD-DC); and (3) mutations in other, currently uncharacterized, genes, which result in autosomal recessive DC (AR-DC). As DKC1 encodes Dyskerin, a protein component of small nucleolar ribonucleoprotein (snoRNP) particles, which are important in ribosomal RNA processing, DC was initially described as a disorder of defective ribosomal biogenesis. Subsequently, Dyskerin and TERC were shown to closely associate with each other in the telomerase complex, and DC has since come to be regarded as a telomerase deficiency disorder characterised by shorter telomeres. These findings demonstrate the importance of telomerase in humans and highlight how its deficiency (through DKC1 and TERC mutations) results in multiple abnormalities including premature ageing, bone marrow failure and cancer. Identification of the gene(s) involved in AR-DC will help to define the pathophysiology of DC further, as well as expand our insights into telomere function, ageing and cancer.