Telomerase RNA Component Levels Research Articles

Telomere transcripts act as tumor suppressor and are associated with favorable prognosis in colorectal cancer with low proliferating cell nuclear antigen expression.

Telomeric repeat-containing RNAs (TERRA) and telomerase RNA component (TERC) regulate telomerase activity (TA) and thereby contribute to telomere homeostasis by influencing telomere length (TL) and the cell immortality hallmark of cancer cells. Additionally, the non-canonical functions of telomerase reverse transcriptase (TERT) and TERRA appear to be involved in the epithelial-mesenchymal transition (EMT), which is important for cancer progression. However, the relationship between TERRA and patient prognosis has not been fully characterized. In this small-scale study, 68 patients with colorectal cancer (CRC) were evaluated for correlations between telomere biology, proliferation, and EMT gene transcripts and disease outcome. The proliferating cell nuclear antigen (PCNA) and the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) showed a positive correlation with TERRA, while TA and TERRA exhibited an inverse correlation. Consistent with previous findings, the present study revealed higher expression levels of TERT and TERC, and increased TA and TL in CRC tumor tissue compared to adjacent non-tumor tissue. In contrast, lower expression levels of TERRA were observed in tumor tissue. Patients with high TERRA expression and low PCNA levels exhibited favorable overall survival rates compared to individuals with the inverse pattern. Furthermore, TERRA suppressed CRC tumor growth in severe combined immunodeficiency disease (SCID) mice. In conclusion, our study extends previously published research on TERRA suggesting its potential therapeutic role in telomerase-positive CRC.

Dyskerin and telomerase RNA component are sex-differentially associated with outcomes and Sunitinib response in patients with clear cell renal cell carcinoma

BackgroundClear cell renal cell carcinoma (ccRCC) displays sex-biased incidence, outcomes, molecular alterations and treatment efficacy; however, clinical managements are largely identical in male and female patients. Moreover, many biomarkers have been identified as predictors for ccRCC outcomes and response to therapeutic drugs, such as multitargeted tyrosine-kinase receptor (TKR) inhibitors, but little is known about their sex-specificity. Dyskerin (DKC1), encoded by the DKC1 gene within Xq28, is a telomerase co-factor stabilizing telomerase RNA component (TERC) and overexpressed in various cancers. Here, we determined whether DKC1 and/or TERC affect ccRCC sex-differentially.MethodsDKC1 and TERC expression in primary ccRCC tumors was assessed using RNA sequencing and qPCR. DKC1 association with molecular alterations and overall or progression-free survival (OS or PFS) was analyzed in the TCGA cohort of ccRCC. The IMmotion 151 and 150 ccRCC cohorts were analyzed to evaluate impacts of DKC1 and TERC on Sunitinib response and PFS.ResultsDKC1 and TERC expression was significantly upregulated in ccRCC tumors. High DKC1 expression predicts shorter PFS independently in female but not male patients. Tumors in the female DKC1-high group exhibited more frequent alterations in PIK3CA, MYC and TP53 genes. Analyses of the IMmotion 151 ccRCC cohort treated with the TKR inhibitor Sunitinib showed that female patients in the DKC1-high group was significantly associated with lower response rates (P = 0.021) accompanied by markedly shortened PFS (6.1 vs 14.2 months, P = 0.004). DKC1 and TERC expression correlated positively with each other, and higher TERC expression predicted poor Sunitinib response (P = 0.031) and shorter PFS (P = 0.004), too. However, DKC1 rather than TERC acted as an independent predictor (P < 0.001, HR = 2.0, 95% CI 1.480–2.704). In male patients, DKC1 expression was associated with neither Sunitinib response (P = 0.131) nor PFS (P = 0.184), while higher TERC levels did not predict response rates. Similar results were obtained from the analysis of the Sunitinib-treated IMmotion 150 ccRCC patients.ConclusionsDKC1 serves as an independent female-specific predictor for survival and Sunitinib efficacy in ccRCC, which contribute to better understanding of the sex-biased ccRCC pathogenesis and improve personalized interventions of ccRCC.

Chemical inhibition of PAPD5/7 rescues telomerase function and hematopoiesis in dyskeratosis congenita

AbstractDyskeratosis congenita (DC) is a pediatric bone marrow failure syndrome caused by germline mutations in telomere biology genes. Mutations in DKC1 (the most commonly mutated gene in DC), the 3′ region of TERC, and poly(A)-specific ribonuclease (PARN) cause reduced levels of the telomerase RNA component (TERC) by reducing its stability and accelerating TERC degradation. We have previously shown that depleting wild-type DKC1 levels by RNA interference or expression of the disease-associated A353V mutation in the DKC1 gene leads to decay of TERC, modulated by 3′-end oligoadenylation by noncanonical poly(A) polymerase 5 (PAPD5) followed by 3′ to 5′ degradation by EXOSC10. Furthermore, the constitutive genetic silencing of PAPD5 is sufficient to rescue TERC levels, restore telomerase function, and elongate telomeres in DKC1_A353V mutant human embryonic stem cells (hESCs). Here, we tested a novel PAPD5/7 inhibitor (RG7834), which was originally discovered in screens against hepatitis B viral loads in hepatic cells. We found that treatment with RG7834 rescues TERC levels, restores correct telomerase localization in DKC1 and PARN-depleted cells, and is sufficient to elongate telomeres in DKC1_A353V hESCs. Finally, treatment with RG7834 significantly improved definitive hematopoietic potential from DKC1_A353V hESCs, indicating that the chemical inhibition of PAPD5 is a potential therapy for patients with DC and reduced TERC levels.

Posttranscriptional manipulation of TERC reverses molecular hallmarks of telomere disease.

The telomerase RNA component (TERC) is a critical determinant of cellular self-renewal. Poly(A)-specific ribonuclease (PARN) is required for posttranscriptional maturation of TERC. PARN mutations lead to incomplete 3' end processing and increased destruction of nascent TERC RNA transcripts, resulting in telomerase deficiency and telomere diseases. Here, we determined that overexpression of TERC increased telomere length in PARN-deficient cells and hypothesized that decreasing posttranscriptional 3' oligo-adenylation of TERC would counteract the deleterious effects of PARN mutations. Inhibition of the noncanonical poly(A) polymerase PAP-associated domain-containing 5 (PAPD5) increased TERC levels in PARN-mutant patient cells. PAPD5 inhibition was also associated with increases in TERC stability, telomerase activity, and telomere elongation. Our results demonstrate that manipulating posttranscriptional regulatory pathways may be a potential strategy to reverse the molecular hallmarks of telomere disease.

Mutations in the Poly(A)-Specific Ribonuclease (PARN) Impair Telomerase RNA 3' End Maturation in Dyskeratosis Congenita Patients

Mutations in 11 genes have been described in patients with dyskeratosis congenita (DC) and related telomere diseases, and account for ~60% of cases. Amongst these, loss-of-function mutations in the poly(A)-specific ribonuclease (PARN) have most recently been found in patients with DC and idiopathic pulmonary fibrosis. PARN has no known role in telomere biology. Why PARN deficiency should mimic telomere diseases remains unclear. Based on its 7-methylguanylate (m7 G) cap recognition and poly(A) deadenylase functions, PARN's primary role is considered to be in regulating mRNA metabolism. Recent studies implicate PARN in non-coding RNA biogenesis, including maturation of small nucleolar RNAs (snoRNAs) via the deadenylation of oligo-adenylated (oligo(A)) intermediates. In two DC patients with biallelic defects in the PARN gene, we found decreased levels of the telomerase RNA component (TERC). TERC is the essential non-coding RNA template and scaffold of the telomerase holoenzyme. TERC possesses an m7 G cap but unlike mRNAs does not have a long poly(A) tail; rather, TERC contains a box H/ACA motif as is found in some snoRNAs. We hypothesized that TERC is regulated by PARN in a manner similar to snoRNAs, given their shared 3′ box H/ACA architecture. Here, usingsomatic cells and induced pluripotent stem (iPS) cells from DC patients with PARN mutations, we show that PARN is required for the 3′ end maturation and accumulation of TERC RNA. In PARN-mutant iPS cells and PARN-deficient cell lines, we find impaired telomerase activity and telomere maintenance. Deep sequencing of TERC 3' ends reveals that PARN is required for the removal of genomically-encoded extensions and post-transcriptionally acquired oligo(A) tails that target nuclear RNAs for destruction. In keeping with this, TERC transcripts from PARN-mutant patient cells decay at an accelerated rate. The diminished TERC steady-state levels, increased oligo(A) forms of TERC, and defects in telomere maintenance in patient cells are rescued by ectopic expression of PARN. Global transcript analysis by RNA-Seq in patient cells and PARN-deficient cell lines compared to controls reveals no protein coding mRNAs that consistently manifest a fold-change exceeding the change in TERC levels. Our data indicate that PARN functions in TERC biogenesis via deadenylation of oligo-adenylated nascent transcripts, which promotes 3' end maturation and stability. Given these findings and the phenotype of patients with PARN mutations, we speculate that a major, non-redundant function of PARN in human cells is regulating the maturation of TERC and other non-coding RNAs, more so than mRNA metabolism. Our results reveal a novel role for PARN in the biogenesis of TERC, and provide a mechanism linking PARN mutations to defective telomere maintenance in DC and related diseases. DisclosuresNo relevant conflicts of interest to declare.

Poly(A)-specific ribonuclease (PARN) mediates 3′-end maturation of the telomerase RNA component

Mutations in the PARN gene (encoding poly(A)-specific ribonuclease) cause telomere diseases including familial idiopathic pulmonary fibrosis (IPF) and dyskeratosis congenita, but how PARN deficiency impairs telomere maintenance is unclear. Here, using somatic cells and induced pluripotent stem cells (iPSCs) from patients with dyskeratosis congenita with PARN mutations, we show that PARN is required for the 3'-end maturation of the telomerase RNA component (TERC). Patient-derived cells as well as immortalized cells in which PARN is disrupted show decreased levels of TERC. Deep sequencing of TERC RNA 3' termini shows that PARN is required for removal of post-transcriptionally acquired oligo(A) tails that target nuclear RNAs for degradation. Diminished TERC levels and the increased proportion of oligo(A) forms of TERC are normalized by restoring PARN, which is limiting for TERC maturation in cells. Our results demonstrate a new role for PARN in the biogenesis of TERC and provide a mechanism linking PARN mutations to telomere diseases.

Dyskerin and TERC expression may condition survival in lung cancer patients.

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.

161 MODULATION OF TELOMERASE ACTIVITY IN BOVINE EMBRYOS USING CYTOPLASMATIC PLASMID INJECTION

Telomeres are repetitive, noncoding sequences at the ends of linear chromosomes that shorten with each cell division. They play an important role in aging and affect the regenerative capacity of cells. The holoenzyme telomerase rebuilds telomeres and is composed of 2 components, i.e. the catalytic protein component telomerase reverse transcriptase (TERT) and the telomerase RNA component (TERC). In mammals, telomerase is active during embryogenesis, in germ cells and a subset of stem and progenitor cells. In the present study, we set out to express the TERC component alone and then in combination with TERT, the human telomerase complex, in bovine embryos. The human telomerase components are highly homologous to bovine telomerase genes. Here, 3 different expression constructs encoding hTERC, hTERT, and a green fluorescent protein (GFP) reporter were co-injected into bovine zygotes cytoplasm, and three groups of 528, 1865, and 110 zygotes were constituted; hTERC/GFP (Group 1), hTERT/hTERC/GFP (Group 2), and GFP alone (Group 3), respectively. GFP fluorescence was used to identify successfully injected embryos. This method has recently been established in our laboratory. Injected and control embryos were cultured for 7 days to the blastocyst stage in vitro and the impact on early embryonic development and the physiological consequences of an ectopic overexpression of telomerase in early bovine embryos were assayed. We obtained 45 blastocysts with green fluorescence in the first, 192 in the second, and 28 in the third group. Embryos with GFP fluorescence were frozen for subsequent PCR analysis and telomerase activity measurement. Some blastocyts were analyzed using quantitative fluoresence in situ hybridization to monitor telomere length. Control groups were analyzed for the endogenous levels of TERC and TERT. Results indicate that endogenous TERC and TERT are up-regulated in morulae and blastocyts. In this study, we show that human TERC and TERT can be expressed in blastocysts by cytoplasmic plasmid injection in bovine zygotes. Statistical analyses were performed using the JMP 7.0.1 for Windows software (SAS Institute Inc., Cary, NC, USA). The Tukey-Kramer test was applied to compare the group means. The expression of hTERC alone resulted in a significant extension of telomere length of 280 telomere fluorescence units. Expression of both components also resulted in a significant extension of telomere length. In conclusion, TERC component alone is sufficient to elongate telomere length. The activity measurement showed that telomerase activity in the hTERT and hTERC injected group is 1.77 times higher than in the control group. Findings from this study will allow a comprehensive analysis of the functions of TERT and TERC in early embryogenesis. The ectopic expression of telomerase components in bovine embryos could pave new avenues for generating stem cells and for the development of novel regenerative therapies. Funded by DFG.

Telomere Elongation in Dyskeratosis Congenita Induced Pluripotent Stem Cells.

Abstract 497Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer premature degeneration of multiple tissues. Bone marrow failure is the principal cause of mortality, and allogeneic stem cell transplantation is limited by increased treatment-related mortality. Somatic cells can be reprogrammed using defined genetic and chemical factors, yielding “induced pluripotent stem” (iPS) cell lines which have the capacity to differentiate into any tissue. Patient-specific iPS cells therefore hold promise as therapeutic agents and disease models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, and we have found that telomere length is increased in human iPS cells relative to the normal primary somatic cells from which they are derived. Here we investigated whether defects in telomerase function would limit derivation or self-renewal of iPS cells from patients with DC. We reprogrammed primary fibroblasts from patients with X-linked and autosomal dominant DC, caused by mutations in the genes encoding dyskerin and telomerase RNA component (TERC), respectively. We were able to establish multiple DC-specific iPS lines showing all hallmarks of pluripotency, including the formation of hematopoietic progenitors in vitro. Unexpectedly, DC-specific iPS cells were able to sustain continual proliferation in vitro, in contrast to the premature senescence displayed by the DC fibroblasts. Although early passage DC iPS cells had shorter telomeres than donor fibroblasts, we found that telomere length in DC iPS cells increased with continued passage in culture. To explain this finding, we discovered that steady state levels of TERC, which are critically limiting in several forms of DC, are upregulated in normal and DC iPS cells. We found that TERC upregulation is a feature of the pluripotent state, that the TERC locus is a target of pluripotency-associated transcription factors, and that transcriptional silencing accompanies a 3' deletion at the TERC locus in autosomal dominant DC. Our results demonstrate that reprogramming restores self-renewal capacity in DC cells despite genetic lesions affecting telomerase, and suggest that strategies to enhance endogenous TERC expression may be feasible and therapeutically beneficial in DC patients. The studies demonstrate the value of patient-specific iPS cells for basic and translational discovery, and further the rationale for autologous iPS based cellular therapy of genetic hematologic disorders. Disclosures:Daley:MPM Capital: Consultancy; Solasia: Consultancy; Epizyme: Consultancy; iPierian: Consultancy, Equity Ownership.

Relationship between Dyskerin Expression and Telomerase Activity in Human Breast Cancer

The nucleolar protein dyskerin is involved in the modification of specific uridine residues to pseudouridine on ribosomal and small nuclear RNAs and in the stabilization of the telomerase RNA component (TERC). In this study we investigated for the first time the relationship between dyskerin expression and telomerase activity in a series of 61 primary breast carcinomas. We found that when dyskerin mRNA values were very low the telomerase activity was markedly reduced, independently of the expression of other important components of the telomerase complex such as telomerase reverse transcriptase (TERT). In vitro experiments showed that reduction of dyskerin expression affect telomerase activity through the reduction of TERC. Only when TERC levels were strongly reduced telomerase activity was hindered. Retroviral mediated over-expression of TERC abolished the telomerase impairment due to dyskerin knock down. In conclusion, our results indicated that, beside its effect on ribosome biogenesis, the levels of dyskerin in cancer cells modulate telomerase activity through the regulation of TERC levels, independently of TERT expression. This should be taken into consideration when utilizing TERT expression as a surrogate indicator of telomerase activity in tumour pathology.

Relationship between dyskerin expression and telomerase activity in human breast cancer.

The nucleolar protein dyskerin is involved in the modification of specific uridine residues to pseudouridine on ribosomal and small nuclear RNAs and in the stabilization of the telomerase RNA component (TERC). In this study we investigated for the first time the relationship between dyskerin expression and telomerase activity in a series of 61 primary breast carcinomas. We found that when dyskerin mRNA values were very low the telomerase activity was markedly reduced, independently of the expression of other important components of the telomerase complex such as telomerase reverse transcriptase (TERT). In vitro experiments showed that reduction of dyskerin expression affect telomerase activity through the reduction of TERC. Only when TERC levels were strongly reduced telomerase activity was hindered. Retroviral mediated over-expression of TERC abolished the telomerase impairment due to dyskerin knock down. In conclusion, our results indicated that, beside its effect on ribosome biogenesis, the levels of dyskerin in cancer cells modulate telomerase activity through the regulation of TERC levels, independently of TERT expression. This should be taken into consideration when utilizing TERT expression as a surrogate indicator of telomerase activity in tumour pathology.

Telomerase reverse-transcriptase homozygous mutations in autosomal recessive dyskeratosis congenita and Hoyeraal-Hreidarsson syndrome

Dyskeratosis congenita (DC) is a multisystem bone marrow failure syndrome characterized by a triad of mucocutaneous abnormalities and an increased predisposition to malignancy. X-linked DC is due to mutations in DKC1, while heterozygous mutations in TERC (telomerase RNA component) and TERT (telomerase reverse transcriptase) have been found in autosomal dominant DC. Many patients with DC remain uncharacterized, particularly families displaying autosomal recessive (AR) inheritance. We have now identified novel homozygous TERT mutations in 2 unrelated consanguineous families, where the index cases presented with classical DC or the more severe variant, Hoyeraal-Hreidarsson (HH) syndrome. These TERT mutations resulted in reduced telomerase activity and extremely short telomeres. As these mutations are homozygous, these patients are predicted to have significantly reduced telomerase activity in vivo. Interestingly, in contrast to patients with heterozygous TERT mutations or hemizygous DKC1 mutations, these 2 homozygous TERT patients were observed to have higher-than-expected TERC levels compared with controls. Collectively, the findings from this study demonstrate that homozygous TERT mutations, resulting in a pure but severe telomerase deficiency, produce a phenotype of classical AR-DC and its severe variant, the HH syndrome.

Genetic heterogeneity in autosomal recessive dyskeratosis congenita with one subtype due to mutations in the telomerase-associated protein NOP10

Dyskeratosis congenita (DC) is characterized by multiple features including mucocutaneous abnormalities, bone marrow failure and an increased predisposition to cancer. It exhibits marked clinical and genetic heterogeneity. DKC1 encoding dyskerin, a component of H/ACA small nucleolar ribonucleoprotein (snoRNP) particles is mutated in X-linked recessive DC. Telomerase RNA component (TERC), the RNA component and TERT the enzymatic component of telomerase, are mutated in autosomal dominant DC, suggesting that DC is primarily a disease of defective telomere maintenance. The gene(s) involved in autosomal recessive DC remains elusive. This paper describes studies aimed at defining the genetic basis of AR-DC. Homozygosity mapping in 16 consanguineous families with 25 affected individuals demonstrates that there is no single genetic locus for AR-DC. However, we show that NOP10, a component of H/ACA snoRNP complexes including telomerase is mutated in a large consanguineous family with classical DC. Affected homozygous individuals have significant telomere shortening and reduced TERC levels. While a reduction of TERC levels is not a universal feature of DC, it can be brought about through a reduction of NOP10 transcripts, as demonstrated by siRNA interference studies. A similar reduction in TERC levels is also seen when the mutant NOP10 is expressed in HeLa cells. These findings identify the genetic basis of one subtype of AR-DC being due to the first documented mutations in NOP10. This further strengthens the model that defective telomere maintenance is the primary pathology in DC and substantiates the evidence in humans for the involvement of NOP10 in the telomerase complex and telomere maintenance.