Functional characterization of TERT alternative splice variants
Final Report Abstract
Telomerase is a cellular machine that consists of the catalytic subunit TERT, a protein, and the RNA TER. Telomerase determines how often cells are able to divide by elongating chromosome ends, the telomeres. Consistent with this role, telomerase is upregulated in highly proliferating cells such as cancer cells and embryonic tissues. Human TERT (hTERT) undergoes complex alternative splicing, a process that leads to different mRNAs and subsequently proteins that are derived from the same gene. The beta-deletion hTERT splice variant results from complete skipping of exon 7 and 8, leading to deletion of most of the domain that is necessary for telomerase catalytic activity. I could show that the beta-deletion mRNA is as abundant as the full-length hTERT in a panel of 52 breast cancer cell lines, human embryonic stem cells and normal T-cells. Although this splice variant carries a premature translation stop codon, I could show that it is not subjected to a cellular control mechanism that degrades such transcripts. Although the presence of the beta-deletion transcript has been established in many different cell lines, means for detection and identification of the endogenous protein isoform were not available. I made use of modified bacterial artificial chromosomes (BACs) containing an hTERT gene that carries a fluorescent tag and that, when reintroduced into a human tissue culture cell, allows for preserved endogenous gene regulation and expression of hTERT splice variants. A stable HeLa cell line carrying the beta-deletion variant tagged at its C-terminus has been generated. These cells have fluorescent signals in the nucleus and the mitochondria, thus establishing for the first time that, and where in the cell, an endogenous hTERT splice variant is expressed at the protein level. I could also show that the beta-deletion isoform can bind human TER (hTER), but is not able to prolong telomeres due to its inactive catalytic domain. I currently investigate the cellular role of this isoform. Since the beta deletion isoform is localized to the mitochondria, I plan to study its function in the mitochondria. Because it has been shown that the full-length hTERT can bind mitochondrial RNAs, I will test whether this isoform has the ability to bind and/or is involved in the processing of mitochondrial RNAs.
