Characterization of novel alternative splicing sites in human telomerase reverse transcriptase (hTERT): analysis of expression and mutual correlation in mRNA isoforms from normal and tumour tissues

Abstract

Background: Human telomerase reverse transcriptase (hTERT) is a key component for synthesis and maintenance of telomeres on chromosome ends and is required for the continued proliferation of cells. Estimation of hTERT expression therefore has broad relevance in oncology and stem cell research. Several splicing variants of hTERT have been described whose regulated expression contributes to the control of telomerase activity. Knowledge of the different hTERT mRNA isoforms and the ability to distinguish between them is an important issue when evaluating telomerase expression.

Results: By establishing cDNA-clone panels from lung and colon tissues, we could map hTERT clones individually for differences in DNA sequence. This made possible the identification of novel alternatively spliced sites as well as analysis of their frequency and mutual correlation in mRNA isoforms. Ten different alternatively spliced sites were detected, of which six were novel sites resulting from alternative splicing of intron 2 or 14. The majority of hTERT cDNA clones from normal and tumour lung and colon tissues encoded truncated proteins ending close after exon 2 or 6.

Conclusion: The increased complexity in telomerase expression revealed here has implications for our understanding of telomerase regulation and for the choice of suitable methods for addressing hTERT expression.

Figure 1

Genomic organization and ASPSs of the hTERT gene. (A) Exon/intron organization of the hTERT gene assembled from [Genbank:AF128893] and [GenBank:AF128894] [14]. Exons are shown as black boxes with numbering above. Start/Stop indicates the beginning and end of the ORF. (B) Schematic drawing of hTERT mRNA ASPSs. Exons are shown as open boxes with numbering inside. Black boxes represent intronic sequences and are cross-hatched to indicate uncharacterised 5' or 3' splice site. The ORF is indicated below each ASPS by arrows. The hTERT ASPSs have been assigned descriptive names: Ins-i, insertion of intronic sequence; Del-e, deletion of exonic sequence; followed by intron/exon number and a range of nucleotides in brackets when the insertion/deletion involves part of an intron/exon, respectively. (C) Formerly used designations: *[9], **[14], ***[15]. (D) Sequence of donor and acceptor splice sites. Unspliced sequence is boxed. Range of spliced nucleotides numbered from the first nucleotide of each intron is shown above the sequence.

Figure 2

Frequency of hTERT mRNA ASPSs in RT-PCR cDNA clones. The tissue of origin is indicated above the graph with number of analysed clones in brackets. Black bars show the percentage of clones containing the indicated ASPS. The fraction of these for which the 5'-splice donor site is continuous with the ORF (not masked by other ASPSs further upstream that terminate the ORF) is shown by an adjacent cross-hatched bar.