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Schematic presentation of biology of alternative splicing (AS). (A) Traditional classification of basic types of alternative RNA splicing events. Exons are represented as blue and grey blocks, introns as lines in between. (B) Functioning of spliceosome. Pre-mRNA containing two exons separated by an intron assembles into splicing complexes together with spliceosome subunits. Individual subunits are indicated by U1, U2, U2AF, U4, U5, and U6. U1 forms a base-pairing interaction with 5′-splice site, whereas U2 base-pairs with branch-point and U2AF binds to polypyrimidine site of 3′ splice site. Then, a complex containing U4, U5, and U6 associates with the forming of spliceosome. The intron is removed in a form of lariat and two exons are ligated. (C) Interactions of trans-elements and serine/arginine-rich (SR) proteins with cis-elements, and regulatory sequences on pre-mRNA. Elements that inhibit exon inclusion are shown in red, while those enhancing inclusion are shown in green. ESE, exonic splicing enhancer; ESI, exonic splicing silencer; ISE, intronic splicing enhancer; ISS, intronic splicing silencer.
Source publication
Alternative splicing (AS) of human telomerase catalytic subunit (hTERT, human telomerase reverse transcriptase) pre-mRNA strongly regulates telomerase activity. Several proteins can regulate AS in a cell type-specific manner and determine the functions of cells. In addition to being involved in telomerase activity regulation, AS provides cells with...
Citations
... In contrast, cancer cells have variable strategies to evade this cellular senescence and death by maintaining their telomere lengths [3]. One primary mechanism of telomere lengthening is the activation of telomerase, an enzyme that elongates the repeat sequences to the ends of chromosomes [4,5]. TERT is a catalytic protein subunit of telomerase and plays a crucial role in this process [5,6]. ...
... One primary mechanism of telomere lengthening is the activation of telomerase, an enzyme that elongates the repeat sequences to the ends of chromosomes [4,5]. TERT is a catalytic protein subunit of telomerase and plays a crucial role in this process [5,6]. TERTp mutations lead to enhanced expression and activity of telomerase, thus contributing to cellular immortality, a hallmark of cancer [7][8][9]. ...
Simple Summary
Telomerase reverse transcriptase promoter (TERTp) mutation is commonly observed in brain tumors and are known to contribute to the acquisition of immortality in tumors via maintaining telomere length. In this study, we aimed to investigate the prognostic impact of several known prognostic factors, including TERTp mutations, in 528 adult-type diffuse gliomas classified according to the 2021 WHO criteria. Our data showed that TERTp mutation status had a significant impact on prognosis in the combined group of glioblastoma, IDH-wildtype and astrocytoma, IDH-mutant, but was not a predictor of prognosis within any individual tumor groups. We also showed that several known clinicopathologic factors have different prognostic significance depending on the type of tumor. This supports the need for systematic tumor diagnosis based on molecular pathology classification and indicates that multiple factors, not just TERTp mutation status, should be considered in the prognosis of tumors.
Abstract
Mutation in the telomerase reverse transcriptase promoter (TERTp )is commonly observed in various malignancies, such as central nervous system (CNS) tumors, malignant melanoma, bladder cancer, and thyroid carcinoma. These mutations are recognized as significant poor prognostic factors for these tumors. In this investigation, a total of 528 cases of adult-type diffuse gliomas diagnosed at a single institution were reclassified according to the 2021 WHO classifications of CNS tumors, 5th edition (WHO2021). The study analyzed clinicopathological and genetic features, including TERTp mutations in each tumor. The impact of known prognostic factors on patient outcomes was analyzed through Kaplan–Meier survival and Cox regression analysis. TERTp mutations were predominantly identified in 94.1% of oligodendrogliomas (ODG), followed by 66.3% in glioblastoma, IDH-wildtype (GBM-IDHwt), and 9.2% of astrocytomas, IDH-mutant (A-IDHm). When considering A-IDHm and GBM as astrocytic tumors (Group 1) and ODGs (Group 2), TERTp mutations emerged as a significant adverse prognostic factor (p = 0.013) in Group 1. However, within each GBM-IDHwt and A-IDHm, the presence of TERTp mutations did not significantly impact patient prognosis (p = 0.215 and 0.268, respectively). Due to the high frequency of TERTp mutations in Group 2 (ODG) and their consistent prolonged survival, a statistical analysis to evaluate their impact on overall survival was deemed impractical. When considering MGMTp status, the combined TERTp-mutated and MGMTp-unmethylated group exhibited the worst prognosis in OS (p = 0.018) and PFS (p = 0.034) of GBM. This study confirmed that the classification of tumors according to the WHO2021 criteria effectively reflected prognosis. Both uni- and multivariate analyses in GBM, age, MGMTp methylation, and CDKN2A/B homozygous deletion were statistically significant prognostic factors while in univariate analysis in A-IDHm, grade 4, the Ki-67 index and MYCN amplifications were statistically significant prognostic factors. This study suggests that it is important to classify and manage tumors based on their genetic characteristics in adult-type diffuse gliomas.
... Human telomerase reverse transcriptase (hTERT) is a crucial enzyme involved in preserving telomeres, which are essential structures found at the ends of chromosomes [168]. ...
Non-melanoma skin cancer (NMSC) encompasses various skin malignancies, primarily basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Extended exposure to environmental elements, particularly solar ultraviolet (UV) radiation, triggers oxidative stress in skin tissues, resulting in DNA damage that is instrumental in the initiation and progression of NMSC. The imbalance between pro-inflammatory and anti-inflammatory cytokines plays a significant role in the development and progression of skin cancer. Resveratrol (RES), an organic phytoalexin present in grape skins and seeds, demonstrates promising chemopreventive and anti-neoplastic capabilities against NMSC. This research revealed that the synergistic lipid-nanocarrier incorporating RES and 5-FU showed significantly enhanced effectiveness in curtailing the proliferation of malignant cells, notably in the A431 cell line, in comparison to traditional formulations. Moreover, the study illustrated that the RES and 5-FU amalgamation exerted a combined effect in hindering cellular proliferation and triggering apoptosis in cancerous cells, as evidenced in both in vitro and in vivo experiments. Furthermore, RES is known for its antioxidant attributes, which may alleviate the impact of ROS triggered by UV exposure, thus diminishing DNA impairment and mutations. These observations imply that RES might offer chemopreventive benefits for NMSC through its role in apoptosis and mitigating oxidative stress. This research offers critical insights into the prospective utility of RES as a safer, more effective intervention for NMSC, though additional investigations are required to thoroughly decipher the underlying molecular mechanisms and clinical implications.
... In a different study, advanced stages of endometrial cancer and colonic adenocarcinoma were found to have high expression of the synthetic subunit with TRTase activity. These results demonstrate a high level of neoplasia that could be employed as a predictive biomarker to discover malignant tumors with a high incidence of recurrence (Plyasova and Zhdanov, 2021). ...
Background: Attributes of potential targets for anticancer therapy are currently being investigated for telomeres, telomerase, and their combined complex.
Study Design: Telomerase (TRTase) is a distinct reverse transcriptase that is thought to be the leader in almost all cancer-derived cells and is primarily in charge of controlling telomere (TLM) length. This enzyme therefore guarantees unabated cell division during carcinogenesis, a characteristic of cancer, and this distinguishing factor has made TRTase the spotlight target for pharmaceutical research in cancer therapy. The capacity of nutraceuticals to inhibit TRTase, impair TLMs, and hinder their complex formation offers a window of opportunity for the discovery of new cancer-treatment targets.
Purpose: This review attempts to provide a fundamental understanding of TLM investigation, TRTase regulatory frameworks, and the diverse binding proteins that hinder the TLM-TRTase combined structure. Moreover, this study highlights nutraceuticals that address TLMs, inhibition at the molecular level, and inhibitors of the catalytic TRTase subunits.
Conclusion: This thorough knowledge of TRTase's physiological action will offer crucial information for boosting the effectiveness of plausible chemotherapeutic drug designs.
... Consequently, cells expressing the α-isoform exhibit reduced telomerase activity and shorter telomeres. This truncated variant is abundant in cancer cells [73] and in activated lymphocytes [74]. Combinations of other splice variants (α + β − and α − β +) have been described in different cell types with a 1-15% range [73]. ...
... This truncated variant is abundant in cancer cells [73] and in activated lymphocytes [74]. Combinations of other splice variants (α + β − and α − β +) have been described in different cell types with a 1-15% range [73]. ...
Telomerase can overcome replicative senescence by elongation of telomeres but is also a specific element in most cancer cells. It is expressed more vastly than any other tumor marker. Telomerase as a tumor target inducing replicative immortality can be overcome by only one other mechanism: alternative lengthening of telomeres (ALT). This limits the probability to develop resistance to treatments. Moreover, telomerase inhibition offers some degree of specificity with a low risk of toxicity in normal cells. Nevertheless, only one telomerase antagonist reached late preclinical studies. The underlying causes, the pitfalls of telomerase-based therapies, and future chances based on recent technical advancements are summarized in this review. Based on new findings and approaches, we propose a concept how long-term survival in telomerase-based cancer therapies can be significantly improved: the TICCA (Transient Immediate Complete and Combinatory Attack) strategy.
... Unlike T cells, B cells do not show telomere erosion upon rapid proliferation, and telomere length in memory B cells is comparable to naïve B cells [163]. This is mainly due to stable expression of telomerase in B cells. ...
Aging attenuates the overall responsiveness of the immune system to eradicate pathogens. The increased production of pro-inflammatory cytokines by innate immune cells under basal conditions, termed inflammaging, contributes to impaired innate immune responsiveness towards pathogen-mediated stimulation and limits antigen-presenting activity. Adaptive immune responses are attenuated as well due to lowered numbers of naïve lymphocytes and their impaired responsiveness towards antigen-specific stimulation. Additionally, the numbers of immunoregulatory cell types, comprising regulatory T cells and myeloid-derived suppressor cells, that inhibit the activity of innate and adaptive immune cells are elevated. This review aims to summarize our knowledge on the cellular and molecular causes of immunosenescence while also taking into account senescence effects that constitute immune evasion mechanisms in the case of chronic viral infections and cancer. For tumor therapy numerous nanoformulated drugs have been developed to overcome poor solubility of compounds and to enable cell-directed delivery in order to restore immune functions, e.g., by addressing dysregulated signaling pathways. Further, nanovaccines which efficiently address antigen-presenting cells to mount sustained anti-tumor immune responses have been clinically evaluated. Further, senolytics that selectively deplete senescent cells are being tested in a number of clinical trials. Here we discuss the potential use of such drugs to improve anti-aging therapy.
... However, several mechanisms for activating telomerase activity have been proposed including deletions/mutations in the TERT promoter, amplification of the TERT gene, alternative splicing of TERT, and epigenetic changes [51]. The role of hTERT transcriptional control and alternative-splicing of hTERT protein has been reported [57][58][59][60] such as 2 deletions (a and ß) and 4 insertions of intron sequences into hTERT mRNA [57,58]. Although the hTERT splice variants are inactive, hTERTa has been reported to be a dominant-negative inhibitor of telomerase, suggesting that splicing can regulate active enzyme levels in cells by altering the balance between the hTERTa form and the full-length transcript [57,58]. ...
Telomere is the repetitive sequence of non-coding DNA that protects chromosomes from damage. However, with cell division, the length of the telomere gets shortened ultimately leading to cell senescence. Telomere shortening is compensated by the addition of telomeric sequence by telomerase enzyme and thus preventing senescence which may lead to abnormal cell growth and ultimately result in cancer. There might not be a direct effect of telomerase on carcinogenesis, however, the role of telomerase in maintaining the length of telomere and thus tumor growth progression is quite evident. Various studies have reported the significance of telomerase activity in tumor cells. Therefore, targeting the telomerase enzyme can be an effective approach for the management of cancer, and drugs targeting telomerase inhibition are possible therapeutic candidates to be used clinically for the treatment of cancer in the future. Thus, in the current paper, we aim to review various telomerase inhibitors against cancer, challenges in proposing telomerase inhibitors for the treatment of cancer, and future perspectives on developing telomerase inhibitors for the management of cancer.
... These telomere-independent functions are considered as telomerase non-canonical functions [8]. Thus, the regulation of the hTERT complex in cancer cells is highly controlled, involving numerous steps such as transcriptional and post-transcriptional mechanisms [9][10][11]. ...
... At the post-transcriptional level, hTERT pre-mRNA is subject to alternative splicing, which generates proteome diversity with different biological functions. To date, around 20 transcript variants have been identified [11,17,18]. Cells exhibiting telomerase activity co-express, at significant levels, different hTERT transcripts and evidence exists that hTERT alternative splicing may play a critical role in the regulation of telomerase activity [19][20][21]. ...
... Aside the telomerase canonical function on the telomere elongation, telomerase also exhibits other non-canonical functions largely implicated in the initiation and progression of cancer [46,57]. The pre-mRNA alternative splicing of hTERT is one of the mechanisms that, despite regulating the telomerase activity, may also play a role in other cellular functions [11,18]. Herein, we reported, for the first time, that hTERT is subjected to alternative splicing in CTCL cells. ...
As a major cancer hallmark, there is a sustained interest in understanding the telomerase contribution to carcinogenesis in order to therapeutically target this enzyme. This is particularly relevant in primary cutaneous T-cell lymphomas (CTCL), a malignancy showing telomerase dysregulation with few investigative data available. In CTCL, we examined the mechanisms involved in telomerase transcriptional activation and activity regulation. We analyzed 94 CTCL patients from a Franco-Portuguese cohort, as well as 8 cell lines, in comparison to 101 healthy controls. Our results showed that not only polymorphisms (SNPs) located at the promoter of human telomerase reverse transcriptase (hTERT) gene (rs2735940 and rs2853672) but also an SNP located within the coding region (rs2853676) could influence CTCL occurrence. Furthermore, our results sustained that the post-transcriptional regulation of hTERT contributes to CTCL lymphomagenesis. Indeed, CTCL cells present a different pattern of hTERT spliced transcripts distribution from the controls, mostly marked by an increase in the hTERT β+ variants proportion. This increase seems to be associated with CTCL development and progression. Through hTERT splicing transcriptome modulation with shRNAs, we observed that the decrease in the α-β+ transcript induced a decrease in the cell proliferation and tumorigenic capacities of T-MF cells in vitro. Taken together, our data highlight the major role of post-transcriptional mechanisms regulating telomerase non canonical functions in CTCL and suggest a new potential role for the α-β+ hTERT transcript variant.
... Moreover, regulatory T cells can inhibit proliferation of T and B lymphocytes as well as natural killer cells in a contact-independent process which involves activation of hTERT alternative splicing [82]. ...
Naturally, in somatic cells chromosome ends (telomeres) shorten during each cell division. This process ensures to limit proliferation of somatic cells to avoid malignant proliferation; however, it leads to proliferative senescence. Telomerase contains the reverse transcriptase TERT, which together with the TERC component, is responsible for protection of genome integrity by preventing shortening of telomeres through adding repetitive sequences. In addition, telomerase has non-telomeric function and supports growth factor independent growth. Unlike somatic cells, telomerase is detectable in stem cells, germ line cells, and cancer cells to support self-renewal and expansion. Elevated telomerase activity is reported in almost all of human cancers. Increased expression of hTERT gene or its reactivation is required for limitless cellular proliferation in immortal malignant cells. In hormonally regulated tissues as well as in prostate, breast and endometrial cancers, telomerase activity and hTERT expression are under control of steroid sex hormones and growth factors. Also, a number of hormones and growth factors are known to play a role in the carcinogenesis via regulation of hTERT levels or telomerase activity. Understanding the role of hormones in interaction with telomerase may help finding therapeutical targets for anticancer strategies. In this review, we outline the roles and functions of several steroid hormones and growth factors in telomerase regulation, particularly in hormone regulated cancers such as prostate, breast and endometrial cancer.
... In some studies, it was shown that apoptotic endonuclease G (EndoG) can produce splice-switching oligonucleotides that act very similarly to circRNA. However, such nucleotides were disrobed for human Telomerase Reverse Transcriptase (hTERT) and Deoxiribonuclease I (DNase I) [63][64][65][66]. Whether EndoG plays a role in the production of circRNAs or not needs to be further studied. ...
Circular RNAs (circRNAs), a novel type of endogenous RNAs, have become a subject of intensive research. It has been found that circRNAs are important players in cell differentiation and tissue homeostasis, as well as disease development. Moreover, the expression of circRNAs is usually not correlated with their parental gene expression, indicating that they are not only a steady-state by-product of mRNA splicing but a product of variable splicing under novel regulation. Sequence conservation analysis has also demonstrated that circRNAs have important non-coding functions. CircRNAs exist as a covalently closed loop form in mammalian cells, where they regulate cellular transcription and translation processes. CircRNAs are built from pre-messenger RNAs, and their biogenesis involves back-splicing, which is catalyzed by spliceosomes. The splicing reaction gives rise to three different types of intronic, exotic and exon–intron circular RNAs. Due to higher nuclease stability and longer half lives in cells, circRNAs are more stable than linear RNAs and have enormous clinical advantage for use as diagnostic and therapeutic biomarkers for disease. In recent years, it has been reported that circRNAs in stem cells play a crucial role in stem cell function. In this article, we reviewed the general feature of circRNAs and the distinct roles of circRNAs in stem cell biology, including regulation of stem cell self-renewal and differentiation. CircRNAs have shown unique expression profiles during differentiation of stem cells and could serve as promising biomarkers of these cells. As circRNAs play pivotal roles in stem cell regulation as well as the development and progression of various diseases, we also discuss opportunities and challenges of circRNA-based treatment strategies in future effective therapies for promising clinical applications.
... To explore whether these effects were still present when targeting endogenous hTERT RNA, further experiments were carried out. The region on hTERT RNA targeted was the alpha region which is present on full length hTERT RNA that is required for telomerase activity [213] , ...
... whereas it is not present in normal tissue and non-cancer cell lines [31,32] , such as BJ fibroblasts, which contain the ∆4-13 variant. HeLa cells have been shown to be telomerase-dependent and naturally contain the full-length hTERT RNA which allows them to avoid cellular senescence and maintain a cancerous phenotype [31,32,203,213,214] . They also contain other hTERT variants lacking the alpha and beta regions of hTERT (absent in normal cells), however variants containing the alpha region are the most prominent [32] making the alpha region a better target. ...
Telomerase hTERT RNA is overexpressed in around 90% of all cancers but targeting it has been unsuccessful to date due to the inability of this approach to kill telomerase-expressing cells leading to the evolution of telomerase-independent cells. The approach proposed in the thesis (NASPER), aims to target cells overexpressing hTERT RNA and cause their apoptosis, preventing this evolution and debulking the tumour mass. NASPER involves bringing two fusion proteins, each of which comprises a custom-designed PUF (Pumilio and FBF) RNA-binding protein and a protease, onto the hTERT RNA into close proximity to activate the protease which should lead to cell death. The proteases tested in this study are the HIV protease (HIVPR) and the split-TEV protease, which require dimerisation/fragment complementation for catalytic activity. In Chapter 3, the two designed PUF proteins were first purified and tested in vitro using fluorescence polarisation experiments to assess RNA binding. The results indicate that the PUF proteins bind specifically to their cognate sequences both independently and in combination. In Chapter 4, expression in E. coli and purification of HIVPR was optimised, and its activity was confirmed in vitro. In cells, the individual PUF-HIVPR fusion proteins appeared to auto-activate without their dimerisation partner despite the use of mutations to reduce auto-activity, and it was concluded that a split system is required such that the protease can only undergo activation when both proteins are present. In Chapter 6, a split-GFP system (fusing two GFP fragments to each of the two PUF proteins) most notably show that, as intended, the fusion proteins bind better when cognate RNA is used versus scrambled RNA, and that engineering the fusion proteins such that the split-GFP are domains oriented towards each other results in improved re-constitution of GFP. Subsequently, a NASPER system designed with the split-TEV protease was tested, in which two TEV protease fragments are fused to each of the two PUF domains. The results were consistent with the split-GFP findings, and NASPER was also able to target overexpressed hTERT mRNA in HeLa cells. No cleavage of a procaspase-3 construct could be detected in apoptosis assays in Chapter 8, and further work is required to elucidate the cause. Chapter 9 describes further experiments to understand the binding characteristics of designed PUF domains using crosslinked RNA-seq methods. Overall, the results presented in the thesis provide new insights into the behaviour of designed PUF proteins in the cell and lay the groundwork for a new therapeutic approach based on targeted protease-induced cell death, as discussed in Chapter 10.
