Canis familiaris telomerase reverse transcriptase undergoes alternative splicing

Abstract

The enzyme telomerase is essential for cell proliferation and tumorigenesis. Telomerase reverse transcriptase (TERT) represents the catalytic subunit of the enzyme. In humans, TERT expression is regulated by several different mechanisms, including alternative splicing. Canis familiaris TERT (dogTERT) has been shown to have a high level of sequence similarity with human TERT, indicating that the dog may represent a suitable animal model for telomerase studies. In the present report we sought to investigate whether dogTERT undergoes alternative splicing. During the analysis of canine mammary tissues (both tumor and paired adjacent to the tumor normal tissues) for dogTERT expression by RT-PCR, we identified eight samples-one tumor and seven adjacent normal-which gave PCR products of unexpected sizes. DNA sequencing revealed two insertions (175 and 28 bp long) and two deletions (17 and 32 bp long), which were encountered in different combinations and gave rise to five different transcripts. The generation of all variants could be explained by the employment of alternative splicing sites within dogTERT genomic sequences. The 175-bp and 28-bp insertions, identified between exons 7 and 8 and between 8 and 9, respectively, constituted unspliced sequences of introns 7 and 8, respectively. Both deletions originated from exon 8 sequence removals due to alternative splicing. All five variants encoded truncated proteins, which lacked essential motifs for reverse transcription and might have thus lost their ability to compose active telomerase enzymes. This is the first identification of alternative splicing events within dogTERT. The results presented here may provide the basis for more thorough studies on the regulation of telomerase activity in canine normal and cancer cells.

Structure—function relationships in telomerase genes

“…Single splicing events (marked by exon or intron number, p stands for partial deletions) of four rat TERT isoforms [deletion del5p, GenBank ® AY539718; insertion i2, no sequence in database; combination i2 + del12p, AY539719; combination i2 + del10, AY539720 (Kaneko et al, 2006)] and one hamster variant [insertion ham i7p (Guo et al, 2001)] are shown above the hTERT variants. Single splicing events of five canine TERT isoforms are shown above the rodent variants [insertion i7p, EU126894; insertion i8p, EU126891; combinations del8p + i8p, EU126890; del8p' + i7p, EU126892; i7p + i8p, EU126893 (Angelopoulou et al, 2008)]. The numerous splice variants of chicken TERT are not included in this Figure and can be found in Hrdlickova et al (2006) and Chang and Delany (2006).…”

“…Four TERT splicing variants have been described in rats with predominant expression of inactive rTERTb variant in adult brain [(Kaneko et al, 2006), Figure 3)] and one splicing variant was reported for hamster TERT (Guo et al, 2001). Five splicing variants were identified for canine TERT (Angelopoulou et al, 2008), and the pattern of spliced variants is apparently complicated in chicken, where 19 isoforms have been reported (Hrdlickova et al, 2006;Chang and Delany, 2006 . Searches for splicing variants failed in Xenopus and mouse Kaneko et al, 2006) but there were candidate sequences in public databases (Table 2).…”

A Vaccine Targeting Telomerase Enhances Survival of Dogs Affected by B-cell Lymphoma

“…[19][20][21][22][23] Pairwise alignments of TERT protein sequences revealed that human protein shares greater homology with Canis familiaris TERT (dTERT) 24 than with mouse TERT 25 and, as observed in human tumors, dTERT activity contributes to maintenance of telomere length. 21 In contrast, telomere shortening and telomerase activation have only a minor effect on murine tumorigenesis.…”

Alternative Splicing of Human Telomerase Reverse Transcriptase (hTERT) and Its Implications in Physiological and Pathological Processes

“…Although some transcriptional TERT patterns are identical to those in humans, most patterns are species-specific. To date, mRNA splice variants different from hTERT were described for chickens [ 34 ], rats and mice [ 48 ], primates [ 39 ], worms [ 49 ], and dogs [ 50 ], among other species.…”