Telomerase reverse transcriptase genes identified in Tetrahymena thermophila and Oxytricha trifallax

Abstract

Telomerase reverse transcriptase (TERT) has been identified as the catalytic subunit of the chromosome end-replicating enzyme in Euplotes, yeasts, and mammals. However, it was not reported among the protein components of purified Tetrahymena telomerase, the first telomerase identified and the most thoroughly studied. It therefore seemed possible that Tetrahymena used an alternative telomerase that lacked a TERT protein. We now report the cloning and sequencing of a Tetrahymena thermophila gene whose encoded protein has the properties expected for a TERT, including large size (133 kDa), basicity (calculated pI = 10.0), and reverse transcriptase sequence motifs with telomerase-specific features. The expression of mRNA from the Tetrahymena TERT gene increases dramatically at 2-5 h after conjugation, preceding de novo addition of telomeres to macronuclear DNA molecules. We also report the cloning and sequencing of the ortholog from Oxytricha trifallax. The Oxytricha macronuclear TERT gene has no introns, whereas that of Tetrahymena has 18 introns. Sequence comparisons reveal a new amino acid sequence motif (CP), conserved among the ciliated protozoan TERTs, and allow refinement of previously identified motifs. A phylogenetic tree of the known TERTs follows the phylogeny of the organisms in which they are found, consistent with an ancient origin rather than recent transposition. The conservation of TERTs among eukaryotes supports the model that telomerase has a conserved core (TERT plus the RNA subunit), with other subunits of the holoenzyme being more variable among species.

Figure 1

Primary structure of the seven known telomerase RTs. Organisms represented are Tetrahymena thermophila (Tt_TERT; this paper), Oxytricha trifallax (Ot_TERT; this paper), Euplotes aediculatus [Ea_p123; (25)], Saccharomyces cerevisiae [Sc_Est2p; (26)], Schizosaccharomyces pombe [Sp_Trt1p; (20)], Homo sapiens [hTERT; (20)], and Mus musculus [mTERT; (30)]. (A) Colored boxes indicate the locations of RT motifs 1, 2 and A to E (41), telomerase-specific motif T (20), and the new motif found in ciliated protozoa, CP. kDa, molecular mass in kilodaltons; pI, isoelectric point. (B) Multiple sequence alignment of the motif amino acid sequences. Distances (in amino acids) between motifs and to the ends of the protein are shown. A consensus derived from all seven sequences is shown above them. Amino acids are included in the consensus if they appear in at least five sequences and are typed in bold if the remaining amino acids are conservative substitutions. Colored residues are conserved throughout all seven sequences. ∗, Amino acid similarity. A consensus derived from just the three ciliated protozoa sequences also is shown for motif CP. Abbreviations for the amino acids are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. The nucleotide sequences of the T. thermophila and O. trifallax genes have been submitted to GenBank (accession nos. AF062652 and AF060230, respectively).

Figure 2

Location of introns in the TERT genes from S. pombe and T. thermophila. Bars represent ORFs with sequence motifs shown as stippled boxes. The two circled pairs of introns show conserved locations in both genes.

Figure 3

A possible phylogenetic tree of all telomerase reverse transcriptases identified to date. The neighbor-joining distance tree was constructed by alignment of motifs T, 1, 2 and A to E as in Fig. 1 and rooted with the ciliated protozoan branch. The statistical support for each node is indicated as the percentage of 1,000 bootstrap replicates that showed that node.

Figure 4

Expression at the RNA level of T. thermophila telomerase components during vegetative growth, starvation for 14 h, and conjugation for 2–24 h as determined by RT-PCR or Northern hybridization. (A) Tt_TERT mRNA, 20 PCR cycles. (B) Telomerase RNA subunit (TER), 13 PCR cycles. (C) U1 snRNA (49), a control for RNA level and RT-PCR efficiency in the different samples, 13 PCR cycles. (D) Northern hybridization analysis of telomerase RNA, probing for U2 snRNA as an internal standard. (E) Quantitation of TERT mRNA levels (dark columns), divided by the signal obtained for the U1 snRNA and then normalized so that the level in vegetative cells equals 1.0. Each column represents the mean of two separate PCRs, with error bars representing the range of values. Quantitation of telomerase RNA levels as determined by Northern hybridization (striped columns), normalized to U2 snRNA so that the value for vegetative cells equals 1.0. Each column represents the mean of four separate samples, with error bars representing the SEM. (Quantitation of the RT-PCR reaction shown in B gives a similar result, with only the vegetative sample showing a slight difference).