Biochemical and Biophysical Research Communications
Inhibition of telomerase activity by splice-switching oligonucleotides targeting the mRNA of the telomerase catalytic subunit affects proliferation of human CD4+ T lymphocytes
Introduction
Telomerase is a ribonucleoprotein complex, which has unique reverse transcriptase activity with the main function of elongating and maintaining telomeres. Telomerase is active in most types of cancer cells, normal germ cells, stem cells, and activated lymphocytes [1]. Two main components of telomerase, the RNA component (TR) that contains the antisense template for telomere synthesis and the catalytic protein (hTERT), are enough for sufficient activity in cell-free conditions in vitro [2]. In vivo telomerase is associated with multiple regulatory proteins, which might be involved in various telomere-independent intracellular pathways (for review see Refs. [3,4]). Many different levels of regulation of the enzyme activity have been investigated [5]. Alternative splicing (AS) of hTERT mRNA is considered as one of the most precise regulators of telomerase activity in human cells. To date, 22 splice variants of hTERT have been described; however, only full-length hTERT (FL hTERT) mRNA encodes a biochemically functional enzyme [6]. In our previous work [7] we demonstrated that apoptotic endonuclease G (EndoG) could induce the deletion of exons 7 and 8 (β–variant) from hTERT mRNA, leading to a reading frame shift that results in the formation of a premature stop codon in exon 10 and the synthesis of a truncated hTERT protein [8,9]. The β– hTERT splice variant encodes inactive isoform with affected reverse transcription domain which acts as a dominant-negative protein [10,11]. EndoG could induce AS due to its RNase activity and its ability to produce the 48-mer oligonucleotide EGPO (EndoG-produced oligonucleotide) [7]. This oligonucleotide pairs with the junction site of exon 8 and intron 8 of hTERT pre-mRNA, and could induce AS in both living cells and naked cell nuclei. The action of EGPO was similar to splice-switching oligonucleotides (SSOs), which are considered a promising instrument for manipulation of protein functions [12,13].
EGPO covers two regulatory sites that are located in intron 8 of hTERT pre-mRNA, UCAUC and ACGGG, which are binding sites for SRp20 and SRp40 splicing regulator proteins, respectively [14]. We believe that base-pairing of specific oligonucleotide with pre-mRNA of hTERT can block the binding of SRp20 and SRp40 proteins to their sites, which results in the induction of the β– splice variant. The aim of this study was to investigate the ability of SSOs, which are complimentary to SRp20 or SRp40 binding sites, to induce AS of hTERT, inhibit telomerase activity and influence human lymphocytes proliferation.
Section snippets
Cell purification and cultivation
All procedures performed in these studies that involved human participants were in accordance with the ethical standards of the institutional and/or national research committee, and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. This study was approved by the Ethical Committee at the Institute of Biomedical Chemistry; written informed consent was obtained from all participants. Blood from healthy 25–35-year-old donors (n = 4) was collected in
Cell transfection with SSO for hTERT pre-mRNA results in telomerase inhibition
In attempt to induce AS of hTERT, we transfected CD4+ T lymphocytes with one of the SSOs, which are complimentary to binding sites of the splicing regulator proteins SRp20 and SRp40 (Fig. 1A–D). Twenty-four hours post transfection, the mRNA levels of hTERT splice variants and telomerase activity were measured in transfected cells. We observed non-significant changes in total hTERT mRNA levels in cell groups transfected with each of single SSO (Fig. 1E). SRp20 24-mer SSO, which blocks only the
Discussion
Functional hTERT is detected in multiple normal somatic human cells, including lymphocytes, brain, liver, prostate, heart, and primary fibroblasts, irrespective of the degree of telomerase activity [21], which denotes its extratelomeric functions. However, the presence of a full-length hTERT variant may not be sufficient to allow hTERT activity when an abundance of spliced variants are concomitantly present. The physiological and pathological significance of hTERT splicing patterns are being
Conflicts of interest
Authors declare no conflicts of interest.
Acknowledgements
The work was performed in the framework of the Program for Basic Research of the State Academies of Sciences for 2013–2020.
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