Dyskeratosis congenita: telomerase, telomeres and anticipation

Dyskeratosis congenita (DC) is a rare bone marrow failure syndrome that displays marked clinical and genetic heterogeneity. The identification of dyskeratosis congenita gene 1 (DKC1) mutations in X-linked recessive patients initially suggested that DC is a defective pseudouridylation disorder. The subsequent identification of mutations in the telomerase RNA component (TERC) of autosomal dominant DC patients together with the discovery that both TERC and the DKC1-encoded protein, dyskerin, are closely associated in the telomerase complex have suggested that the pathophysiology of DC predominantly relates to defective telomere maintenance. Recent discoveries have shown that autosomal dominant DC exhibits disease anticipation and that this is associated with progressive telomere shortening owing to the haplo-insufficiency of TERC.     

Expression of the Genetic Suppressor Element 24.2 (GSE24.2) Decreases DNA Damage and Oxidative Stress in X-Linked Dyskeratosis Congenita Cells

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 ({"type":"entrez-geo","attrs":{"text":"GSE24","term_id":"24"}}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 {"type":"entrez-geo","attrs":{"text":"GSE24","term_id":"24"}}GSE24.2 rescues both global and telomeric DNA damage. Furthermore, transfection of bacterial purified or a chemically synthesized {"type":"entrez-geo","attrs":{"text":"GSE24","term_id":"24"}}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 {"type":"entrez-geo","attrs":{"text":"GSE24","term_id":"24"}}GSE24.2 was able to diminish it. Altogether our data indicated that supplying {"type":"entrez-geo","attrs":{"text":"GSE24","term_id":"24"}}GSE24.2, either from a cDNA vector or as a peptide reduces the pathogenic effects of Dkc1 mutations and suggests a novel therapeutic approach.     

TERC suppresses PD-L1 expression by downregulating RNA binding protein HuR

TERC is the RNA component of telomerase, and provides a template for TERT to synthesize telomere repeats at chromosome ends. Increasing evidence has revealed that TERC is involved in other biological processes beyond telomerase. Here, we found that the expression level of TERC is negatively correlated with PD-L1 and that ectopic expression of TERC but not TERT in ALT cells significantly inhibits PD-L1, suggesting that TERC suppresses PD-L1 expression in a telomerase-independent manner. Mechanistically, instead of regulating PD-L1 mRNA directly, TERC accelerates PD-L1 mRNA degradation by inhibiting the expression of HuR, which binds to the 3′UTR of PD-L1 mRNA and maintains its stability. We also found that the small molecule AS1842856, a FoxO1 inhibitor, promotes TERC expression and reverses the PD-L1 upregulation caused by chemotherapy, providing a potential combination cancer therapy that avoids cancer immune escape during chemotherapy.

     

Dyskerin is a component of the Arabidopsis telomerase RNP required for telomere maintenance

Dyskerin binds the H/ACA box of human telomerase RNA and is a core telomerase subunit required for RNP biogenesis and enzyme function in vivo. Missense mutations in dyskerin result in dyskeratosis congenita, a complex syndrome characterized by bone marrow failure, telomerase enzyme deficiency, and progressive telomere shortening. Here we demonstrate that dyskerin also contributes to telomere maintenance in Arabidopsis thaliana. We report that both AtNAP57, the Arabidopsis dyskerin homolog, and AtTERT, the telomerase catalytic subunit, accumulate in the plant nucleolus, and AtNAP57 associates with active telomerase RNP particles in an RNA-dependent manner. Furthermore, AtNAP57 interacts in vitro with AtPOT1a, a novel component of Arabidopsis telomerase. Although a null mutation in AtNAP57 is lethal, AtNAP57, like AtTERT, is not haploinsufficient for telomere maintenance in Arabidopsis. However, introduction of an AtNAP57 allele containing a T66A mutation decreased telomerase activity in vitro, disrupted telomere length regulation on individual chromosome ends in vivo, and established a new, shorter telomere length set point. These results imply that T66A NAP57 behaves as a dominant-negative inhibitor of telomerase. We conclude that dyskerin is a conserved component of the telomerase RNP complex in higher eukaryotes that is required for maximal enzyme activity in vivo.     

Dyskeratosis congenita: molecular insights into telomerase function, ageing and cancer

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.     

Telomerase dysfunction and dyskeratosis congenita

Dyskeratosis congenita (DC) is a multi system bone marrow failure syndrome characterized by muco-cutaneous abnormalities and an increased predisposition to malignancy. It exhibits considerable clinical and genetic heterogeneity. X-linked recessive, autosomal dominant and autosomal recessive forms of the disease are recognized. The X-linked recessive form is due to mutations in dyskerin, which is a component of both small nucleolar ribonuclear protein particles and the telomerase complex. Autosomal dominant DC is due to mutations in the RNA component of telomerase, TERC. As dyskerin and TERC are both components of the telomerase complex and all patients with DC have short telomeres it appears that the principal pathology in DC relates to telomerase dysfunction. The gene or genes involved in the recessive form of DC remain elusive, though genes whose products are required for telomere maintenance remain strong candidates. The study of DC has highlighted the critical role of telomerase and the consequences, including premature aging and malignancy, of its dysfunction.     

Crystal structure of a Cbf5-Nop10-Gar1 complex and implications in RNA-guided pseudouridylation and dyskeratosis congenita

H/ACA RNA-protein complexes, comprised of four proteins and an H/ACA guide RNA, modify ribosomal and small nuclear RNAs. The H/ACA proteins are also essential components of telomerase in mammals. Cbf5 is the H/ACA protein that catalyzes isomerization of uridine to pseudouridine in target RNAs. Mutations in human Cbf5 (dyskerin) lead to dyskeratosis congenita. Here, we describe the 2.1 A crystal structure of a specific complex of three archaeal H/ACA proteins, Cbf5, Nop10, and Gar1. Cbf5 displays structural properties that are unique among known pseudouridine synthases and are consistent with its distinct function in RNA-guided pseudouridylation. We also describe the previously unknown structures of both Nop10 and Gar1 and the structural basis for their essential roles in pseudouridylation. By using information from related structures, we have modeled the entire ribonucleoprotein complex including both guide and substrate RNAs. We have also identified a dyskeratosis congenita mutation cluster site within a modeled dyskerin structure.     

TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita

Patients with dyskeratosis congenita (DC), a heterogeneous inherited bone marrow failure syndrome, have abnormalities in telomere biology, including very short telomeres and germline mutations in DKC1, TERC, TERT, or NOP10, but approximately 60% of DC patients lack an identifiable mutation. With the very short telomere phenotype and a highly penetrant, rare disease model, a linkage scan was performed on a family with autosomal-dominant DC and no mutations in DKCI, TERC, or TERT. Evidence favoring linkage was found at 2p24 and 14q11.2, and this led to the identification of TINF2 (14q11.2) mutations, K280E, in the proband and her five affected relatives and TINF2 R282H in three additional unrelated DC probands, including one with Revesz syndrome; a fifth DC proband had a R282S mutation. TINF2 mutations were not present in unaffected relatives, DC probands with mutations in DKC1, TERC, or TERT or 298 control subjects. We demonstrate that a fifth gene, TINF2, is mutated in classical DC and, for the first time, in Revesz syndrome. This represents the first shelterin complex mutation linked to human disease and confirms the role of very short telomeres as a diagnostic test for DC.     

Dyskeratosis congenita -- a disease of dysfunctional telomere maintenance

Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome associated with abnormalities of the skin, fingernails, and tongue. Other clinical manifestations may include epiphora, lung fibrosis, liver cirrhosis, osteoporosis, and a predisposition to develop a variety of malignancies. The clinical picture often resembles that of a premature aging syndrome and tissues affected are those with a high cell turnover. DC has been linked to mutations in at least four distinct genes, three of which have been identified. The product of these genes, dyskerin, the telomerase RNA (TERC), and the catalytic unit of telomerase (TERT) are part of a ribonucleoprotein complex, the telomerase enzyme, that is essential for the elongation and maintenance of chromosome ends or telomeres. All patients with DC have excessively short telomeres, indicating that the underlying defect in these individuals is an inability to maintain the telomeres. The purpose of the current review is to highlight recent insights into the molecular pathogenesis of DC. We discuss the impact these findings have on our current understanding of telomere function and maintenance, and on the diagnosis, management, and treatment of patients with conditions caused by dysfunctional telomeres.     

Anomalous electrophoretic migration of newly synthesized ribosomal RNAs and their precursors from cells with DKC1 mutations

Mutations in the X-linked gene, DKC1, encoding dyskerin, cause dyskeratosis congenita by leading to decreased telomerase activity and causing short telomeres. Dyskerin is also a pseudouridine synthase that modifies nascent ribosomal and other RNAs and it is not known if this function is affected by the mutations. Here we show that newly synthesized ribosomal RNA, extracted from human and mouse cells with pathogenic mutations, shows anomalous mobility in agarose gels under certain denaturation conditions. The anomalously migrating RNA is turned over rapidly. Analysis of ribosomal RNA in these cells suggests the altered mobility is due to inefficient pseudouridylation.     

Mitochondrial Telomerase Protects Cancer Cells from Nuclear DNA Damage and Apoptosis

Most cancer cells express high levels of telomerase and proliferate indefinitely. In addition to its telomere maintenance function, telomerase also has a pro-survival function resulting in an increased resistance against DNA damage and decreased apoptosis induction. However, the molecular mechanisms for this protective function remain elusive and it is unclear whether it is connected to telomere maintenance or is rather a non-telomeric function of the telomerase protein, TERT. It was shown recently that the protein subunit of telomerase can shuttle from the nucleus to the mitochondria upon oxidative stress where it protects mitochondrial function and decreases intracellular oxidative stress. Here we show that endogenous telomerase (TERT protein) shuttles from the nucleus into mitochondria upon oxidative stress in cancer cells and analyzed the nuclear exclusion patterns of endogenous telomerase after treatment with hydrogen peroxide in different cell lines. Cell populations excluded TERT from the nucleus upon oxidative stress in a heterogeneous fashion. We found a significant correlation between nuclear localization of telomerase and high DNA damage, while cells which excluded telomerase from the nucleus displayed no or very low DNA damage. We modeled nuclear and mitochondrial telomerase using organelle specific localization vectors and confirmed that mitochondrial localization of telomerase protects the nucleus from inflicted DNA damage and apoptosis while, in contrast, nuclear localization of telomerase correlated with higher amounts of DNA damage and apoptosis. It is known that nuclear DNA damage can be caused by mitochondrially generated reactive oxygen species (ROS). We demonstrate here that mitochondrial localization of telomerase specifically prevents nuclear DNA damage by decreasing levels of mitochondrial ROS. We suggest that this decrease of oxidative stress might be a possible cause for high stress resistance of cancer cells and could be especially important for cancer stem cells.