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Characterization of novel alternative splicing sites in human telomerase reverse transcriptase (hTERT): analysis of expression and mutual correlation in mRNA isoforms from normal and tumour tissues.

ABSTRACT: BACKGROUND: Human telomerase reverse transcriptase (hTERT) is a key component for synthesis and maintenance of telomeres on chromosome ends and is required for the continued proliferation of cells. Estimation of hTERT expression therefore has broad relevance in oncology and stem cell research. Several splicing variants of hTERT have been described whose regulated expression contributes to the control of telomerase activity. Knowledge of the different hTERT mRNA isoforms and the ability to distinguish between them is an important issue when evaluating telomerase expression. RESULTS: By establishing cDNA-clone panels from lung and colon tissues, we could map hTERT clones individually for differences in DNA sequence. This made possible the identification of novel alternatively spliced sites as well as analysis of their frequency and mutual correlation in mRNA isoforms. Ten different alternatively spliced sites were detected, of which six were novel sites resulting from alternative splicing of intron 2 or 14. The majority of hTERT cDNA clones from normal and tumour lung and colon tissues encoded truncated proteins ending close after exon 2 or 6. CONCLUSION: The increased complexity in telomerase expression revealed here has implications for our understanding of telomerase regulation and for the choice of suitable methods for addressing hTERT expression.

SUBMITTER: Saeboe-Larssen S

PROVIDER: S-EPMC1560392 | biostudies-literature | 2006

REPOSITORIES: biostudies-literature

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Characterization of novel alternative splicing sites in human telomerase reverse transcriptase (hTERT): analysis of expression and mutual correlation in mRNA isoforms from normal and tumour tissues.

Saebøe-Larssen Stein S Fossberg Ellen E Gaudernack Gustav G

BMC molecular biology 20060829

<h4>Background</h4>Human telomerase reverse transcriptase (hTERT) is a key component for synthesis and maintenance of telomeres on chromosome ends and is required for the continued proliferation of cells. Estimation of hTERT expression therefore has broad relevance in oncology and stem cell research. Several splicing variants of hTERT have been described whose regulated expression contributes to the control of telomerase activity. Knowledge of the different hTERT mRNA isoforms and the ability to ...[more]

PMID: 16939641

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Project description:BackgroundTelomerase is a reverse transcriptase that maintains the telomeres of linear chromosomes and preserves genomic integrity. The core components are a catalytic protein subunit, the telomerase reverse transcriptase (TERT), and an RNA subunit, the telomerase RNA (TR). Telomerase is unique in its ability to catalyze processive DNA synthesis, which is facilitated by telomere-specific DNA-binding domains in TERT called anchor sites. A conserved glutamine residue in the TERT N-terminus is important for anchor site interactions in lower eukaryotes. The significance of this residue in higher eukaryotes, however, has not been investigated.Methodology/principal findingsTo understand the significance of this residue in higher eukaryotes, we performed site-directed mutagenesis on human TERT (hTERT) Q169 to create neutral (Q169A), conservative (Q169N), and non-conservative (Q169D) mutant proteins. We show that these mutations severely compromise telomerase activity in vitro and in vivo. The functional defects are not due to abrogated interactions with hTR or telomeric ssDNA. However, substitution of hTERT Q169 dramatically impaired the ability of telomerase to incorporate nucleotides at the second position of the template. Furthermore, Q169 mutagenesis altered the relative strength of hTERT-telomeric ssDNA interactions, which identifies Q169 as a novel residue in hTERT required for optimal primer binding. Proteolysis experiments indicate that Q169 substitution alters the protease-sensitivity of the hTERT N-terminus, indicating that a conformational change in this region of hTERT is likely critical for catalytic function.Conclusions/significanceWe provide the first detailed evidence regarding the biochemical and cellular roles of an evolutionarily-conserved Gln residue in higher eukaryotes. Collectively, our results indicate that Q169 is needed to maintain the hTERT N-terminus in a conformation that is necessary for optimal enzyme-primer interactions and nucleotide incorporation. We show that Q169 is critical for the structure and function of human telomerase, thereby identifying a novel residue in hTERT that may be amenable to therapeutic intervention.

Project description:Esophageal adenocarcinoma exhibits one of the highest mortality rates among all cancer entities. Multimodal therapy strategies have improved patients' survival significantly. However, patients in early stages are currently limited to receiving only local therapies, even though some patients within this group showcase short survival periods. Until now, there has been no widely established clinically used biomarker to detect these high-risk patients. Telomerase reverse transcriptase (TERT), a gene encoding a crucial subunit of the telomerase enzyme, plays a significant role in establishing cancer cell immortality and is under suspicion for its potential contribution to tumor progression. Therefore, we aimed to evaluate the clinical relevance of the TERT amplification status. We included 643 patients with esophageal adenocarcinoma, who underwent Ivor-Lewis esophagectomy at the University Hospital of Cologne. The TERT amplification status was characterized using fluorescence in situ hybridization. Clinicopathological values and patients' overall survival were compared between patients with and without TERT amplification. Further sub-cohort analyses were conducted for patients with pT1N0-3 tumor stage. Eighty-One patients (12.6%) exhibited TERT amplification. Patients with amplified TERT showed significantly worse overall survival (median OS: 22.6 vs. 36.8 months, p = 0.009). Interestingly, TERT amplification could be characterized as an independent risk factor for worse overall survival in multivariate analysis in patients with pT1N0-3 tumor stage (HR = 2.440, 95% CI 1.095-5.440, p = 0.029). In this study, we describe the TERT amplification status as an independent risk factor for worse survival in patients diagnosed with esophageal adenocarcinoma at pT1N0-3 tumor stage, encompassing cases involving tumor infiltration of the lamina propria, muscularis mucosae, and/or submucosa. Based on our findings, we put forth the proposition that evaluating the TERT amplification status may serve as a valuable tool in identifying a specific subgroup of patients, namely those with TERT amplification and pT1N0-3 tumor-stage esophageal adenocarcinoma. The patients of this subgroup could potentially benefit from enhanced follow-up protocols, more aggressive treatment approaches, or possible targeted TERT inhibition therapies, all aimed at improving their overall clinical outcomes.

Dataset Information

Characterization of novel alternative splicing sites in human telomerase reverse transcriptase (hTERT): analysis of expression and mutual correlation in mRNA isoforms from normal and tumour tissues.

Publications

Characterization of novel alternative splicing sites in human telomerase reverse transcriptase (hTERT): analysis of expression and mutual correlation in mRNA isoforms from normal and tumour tissues.

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