Alternative splicing and nonsense-mediated decay regulate telomerase reverse transcriptase (TERT) expression during virus-induced lymphomagenesis in vivo

Abstract

BackgroundTelomerase activation, a critical step in cell immortalization and oncogenesis, is partly regulated by alternative splicing. In this study, we aimed to use the Marek's disease virus (MDV) T-cell lymphoma model to evaluate TERT regulation by splicing during lymphomagenesis in vivo, from the start point to tumor establishment.ResultsWe first screened cDNA libraries from the chicken MDV lymphoma-derived MSB-1 T- cell line, which we compared with B (DT40) and hepatocyte (LMH) cell lines. The chTERT splicing pattern was cell line-specific, despite similar high levels of telomerase activity. We identified 27 alternative transcripts of chicken TERT (chTERT). Five were in-frame alternative transcripts without in vitro telomerase activity in the presence of viral or chicken telomerase RNA (vTR or chTR), unlike the full-length transcript. Nineteen of the 22 transcripts with a premature termination codon (PTC) harbored a PTC more than 50 nucleotides upstream from the 3' splice junction, and were therefore predicted targets for nonsense-mediated decay (NMD). The major PTC-containing alternatively spliced form identified in MSB1 (ie10) was targeted to the NMD pathway, as demonstrated by UPF1 silencing. We then studied three splicing events separately, and the balance between in-frame alternative splice variants (d5f and d10f) plus the NMD target i10ec and constitutively spliced chTERT transcripts during lymphomagenesis induced by MDV indicated that basal telomerase activity in normal T cells was associated with a high proportion of in-frame non functional isoforms and a low proportion of constitutively spliced chTERT. Telomerase upregulation depended on an increase in active constitutively spliced chTERT levels and coincided with a switch in alternative splicing from an in-frame variant to NMD-targeted variants.ConclusionsTERT regulation by splicing plays a key role in telomerase upregulation during lymphomagenesis, through the sophisticated control of constitutive and alternative splicing. Using the MDV T-cell lymphoma model, we identified a chTERT splice variant as a new NMD target.

The Regulation of Telomerase by Alternative Splicing of TERT

“…In contrast, only three alternatively spliced variants were identified in quail despite screening various A large number of single AS event variants of avian TERT were identified. Many of the chicken variants were described previously (Amor et al, 2010;Chang & Delany, 2006;Hrdličková et al, 2006). On the other hand, the more distantly related duck share only variant K with chicken, but five other duck variants have certain structural similarity to chicken AS variants (KB, J2, J3K, S, and P).…”

“…Several AS TERT variants have been reported in a number of species including human, rat, dog, chicken, zebrafish, and plants (Sýkorová & Fajkus, 2009). In the human and chicken, which have been extensively studied, 21 and 37 single-event AS TERT variants have been identified (Amor et al, 2010;Chang & Delany, 2006;Hisatomi et al, 2003;Hrdličková et al, 2012a;Hrdličková et al, 2006;Saebøe-Larssen et al, 2006;Wick et al, 1999). Interestingly, none of the human and chicken AS variants are identical, suggesting that AS TERT variants evolved independently in placental mammals and birds.…”

“…The human TERT gene is expressed principally as alternatively spliced (AS) forms in both normal cells and tumor cells (Hisatomi et al, 2003;Hrdličková et al, 2012a;Saebøe-Larssen et al, 2006;Ulaner et al, 1998;Ulaner et al, 2001;Ulaner et al, 2000;Wick et al, 1999). The chicken is the only other species in which alternative splicing of TERT has been extensively characterized and a large number of AS variants have been identified (Amor et al, 2010;Chang & Delany, 2006;Hrdličková et al, 2012a;Hrdličková et al, 2006). AS TERT variants can be divided into two groups -the first group retains the original open reading frame (ORF), while the second group of transcripts contains premature termination codons (PTCs).…”

TERT biology and function in cancer: beyond immortalisation

“…Interestingly, several splicing variants of TERT were described in human and in chicken (Kilian et al 1997, Wick et al 1999, Hisatomi et al 2003, Chang & Delany 2006, Saeboe-Larssen et al 2006, Amor et al 2010. In 2012, another human variant was described, containing an in-frame deletion, which removed exons 4 through 13 (Δ4-13), encoding the catalytic domain of telomerase (Hrdlickova et al 2012).…”

Alternatively Spliced Telomerase Reverse Transcriptase Variants Lacking Telomerase Activity Stimulate Cell Proliferation

“…The other species in which alternative splicing of TERT has been characterized extensively is the chicken. Thirty-one chicken AS TERT variants have previously been identified in various cell lines and tissues (2,11,31). Individual alternative splicing events can occur in various combinations in a single TERT transcript (2,11,13,27,31).…”