Expression Profile of a γ-Deletion Variant of the Human Telomerase Reverse Transcriptase Gene

Higuchi, Hisashi; Ohyashiki, Kazuma; Ohyashiki, Junko H.; Nagao, Kumi; Kanamaru, Taichi; Hirata, Hiroyuki; Hibi, Nozomu; Tsukada, Yutaka

doi:10.1016/s1476-5586(03)80051-9

Cited by 65 publications

(63 citation statements)

References 31 publications

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“…However, in the EWS-FLI1-reduced cells, exon 11 is skipped and the large isoform is significantly reduced. This TERT variant has been classified as the γ-isoform, where the distal reverse transcriptase domain is shortened (68). The only functional data for γ-TERT show that cell lines Huh7 and HLE expressing very small quantities of γ-TERT have 50% greater telomerase activity than cells lacking γ-TERT (68).…”

Section: Yk-4-279 Alters Splicing Rather Than Direct Transcriptional mentioning

confidence: 99%

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

Selvanathan

Graham

Erkizan

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

118

135

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The synthesis and processing of mRNA, from transcription to translation initiation, often requires splicing of intragenic material. The final mRNA composition varies based on proteins that modulate splice site selection. EWS-FLI1 is an Ewing sarcoma (ES) oncoprotein with an interactome that we demonstrate to have multiple partners in spliceosomal complexes. We evaluate the effect of EWS-FLI1 on posttranscriptional gene regulation using both exon array and RNAseq. Genes that potentially regulate oncogenesis, including CLK1, CASP3, PPFIBP1, and TERT, validate as alternatively spliced by EWS-FLI1. In a CLIP-seq experiment, we find that EWS-FLI1 RNA-binding motifs most frequently occur adjacent to intron-exon boundaries. EWS-FLI1 also alters splicing by directly binding to known splicing factors including DDX5, hnRNP K, and PRPF6. Reduction of EWS-FLI1 produces an isoform of γ-TERT that has increased telomerase activity compared with wild-type (WT) TERT. The small molecule YK-4-279 is an inhibitor of EWS-FLI1 oncogenic function that disrupts specific protein interactions, including helicases DDX5 and RNA helicase A (RHA) that alters RNA-splicing ratios. As such, YK-4-279 validates the splicing mechanism of EWS-FLI1, showing alternatively spliced gene patterns that significantly overlap with EWS-FLI1 reduction and WT human mesenchymal stem cells (hMSC). Exon array analysis of 75 ES patient samples shows similar isoform expression patterns to cell line models expressing EWS-FLI1, supporting the clinical relevance of our findings. These experiments establish systemic alternative splicing as an oncogenic process modulated by EWS-FLI1. EWS-FLI1 modulation of mRNA splicing may provide insight into the contribution of splicing toward oncogenesis, and, reciprocally, EWS-FLI1 interactions with splicing proteins may inform the splicing code.T he alternative splicing of mRNA expands the diversity of the human proteome through evolution and ontogeny (1, 2). Spliceosomal network interactions, including proteins that recognize splice enhancer and silencer regions, are critical for the regulation of alternative splicing leading to protein isoforms with disparate functionality (3). Alternative splicing provides both a method by which to categorize subsets of cancers and an avenue for more effective targeted treatments (4). However, the spliceosomal protein interaction networks that are specific to cancer have not been systematically defined; alternative splicing can also change protein-protein interactions within networks (5). A systems biology approach can be used to study the relationship between splicing and oncogenesis by using tumor models with chromosomal translocations whose expressed fusion proteins have putative roles in splicing (6, 7).Fusion proteins produced by chromosomal translocations in sarcomas often contain the amino-terminal portion of EWS and are classified as transcription factors due to the presence of a canonical carboxyl-terminal DNA-binding domain (6). EWS-FLI1 is a well-established ES oncoprotein and is re...

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Section: Yk-4-279 Alters Splicing Rather Than Direct Transcriptional mentioning

confidence: 99%

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

Selvanathan

Graham

Erkizan

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

118

135

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“…20,24 Human telomerase consists of a ribonucleoprotein containing a protein catalytic subunit, the human telomerase reverse transcriptase (hTERT), and an RNA component, the human telomerase RNA, and it may require dimerization or multimerization for optimal activity. 25,26 The hTERT transcript is known to have seven alternative splice sites, which can theoretically produce multiple tissue-and disease-specific alternative transcripts, 27,28 only a few of which have been well documented to date, however. We recently identified four hTERT splice transcript variants in thyroid tumors: full-length, ␣-deletion, ␤-deletion, and ␣-␤-deletion transcripts.…”

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confidence: 99%

Telomere Length Is Related to Alternative Splice Patterns of Telomerase in Thyroid Tumors

Wang

Meeker

Kowalski

et al. 2011

The American Journal of Pathology

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Telomere dysfunction and aberrant telomerase expression play important roles in tumorigenesis. In thyroid tumors, three possibly inhibitory splice variants of the active full-length isoform of human telomerase reverse transcriptase (hTERT) may be expressed. These variants might regulate telomerase activity and telomere length because it is the fraction of the full-length isoform, rather than the total transcript level, that correlates with enzymatic activity. Telomerase reactivation may be critical in the early stages of tumorigenesis, when progressive telomere shortening may be limiting cell viability. The aim of this study was to investigate the relationship between telomere length and hTERT splice variant expression patterns in benign and well-differentiated malignant thyroid tumors. Telomere lengths of 61 thyroid tumors were examined by fluorescence in situ hybridization, comparing tumors with adjacent normal thyroid tissue on the same slide. Expression patterns of hTERT splice variants were evaluated by quantitative and nested RT-PCR. Telomere length was inversely correlated with percentage of full-length hTERT expression rather than with total hTERT expression levels. Short telomeres and high fractions of full-length hTERT transcripts were associated with follicular and papillary thyroid carcinomas, whereas long telomeres and low levels of full-length hTERT were associated with benign thyroid nodules. Intermediate levels of full-length hTERT and telomere length were found in follicular variant of papillary thyroid carcinomas and follicular adenomas.

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“…Several alternatively spliced variants of hTERT have been identified (Kilian et al, 1997;Ulaner et al, 1998;Wick et al, 1999;Yi et al, 2001); one deletion site induces the a-deletion variant, lacking 36 bp from exon 6, and the other induces the b-deletion variant, lacking 182 bp from exons 7 and 8 (Yi et al, 2001). More recently, we have found a new alternatively spliced form, namely the g-deletion variant, lacking the entire exon 11 (Hisatomi et al, 2003;Nagao et al, 2004). Since splicing variants are considered to be nonfunctional forms, it is important to discriminate the fulllength isoform from variants (Yi et al, 2001).…”

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confidence: 99%

Quantitative relationship between functionally active telomerase and major telomerase components (hTERT and hTR) in acute leukaemia cells

Ohyashiki

Higuchi²,

Nagao³

et al. 2005

Br J Cancer

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Functionally active telomerase is affected at various steps including transcriptional and post-transcriptional levels of major telomerase components (hTR and human telomerase reverse transcriptase (hTERT)). We therefore developed a rapid and sensitive method to quantify hTERT and its splicing variants as well as the hTR by a Taqman real-time reverse transcriptase -polymerase chain reaction to determine whether their altered expression may contribute to telomere attrition in vivo or not. Fresh leukaemia cells obtained from 38 consecutive patients were used in this study. The enzymatic level of telomerase activity measured by TRAP assay was generally associated with the copy numbers of full-length hTERT þ a þ b mRNA (P ¼ 0.0024), but did not correlate with hTR expression (P ¼ 0.6753). In spite of high copy numbers of full-length hTERT mRNA, telomerase activity was low in some cases correlating with low copy numbers of hTR, raising the possibility that alteration of the hTR : hTERT ratio may affect functionally active telomerase activity in vivo. The spliced nonactive hTERT mRNA tends to be lower in patients with high telomerase activity, suggesting that this epiphenomenon may play some role in telomerase regulation. An understanding of the complexities of telomerase gene regulation in biologically heterogeneous leukaemia cells may offer new therapeutic approaches to the treatment of acute leukaemia.

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Expression Profile of a γ-Deletion Variant of the Human Telomerase Reverse Transcriptase Gene

Cited by 65 publications

References 31 publications

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

Oncogenic fusion protein EWS-FLI1 is a network hub that regulates alternative splicing

Telomere Length Is Related to Alternative Splice Patterns of Telomerase in Thyroid Tumors

Quantitative relationship between functionally active telomerase and major telomerase components (hTERT and hTR) in acute leukaemia cells

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