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Telomerase Catalytic Subunit Homologs from Fission Yeast and Human

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Science  15 Aug 1997:
Vol. 277, Issue 5328, pp. 955-959
DOI: 10.1126/science.277.5328.955

Abstract

Catalytic protein subunits of telomerase from the ciliateEuplotes aediculatus and the yeast Saccharomyces cerevisiae contain reverse transcriptase motifs. Here the homologous genes from the fission yeast Schizosaccharomyces pombe and human are identified. Disruption of the S. pombe gene resulted in telomere shortening and senescence, and expression of mRNA from the human gene correlated with telomerase activity in cell lines. Sequence comparisons placed the telomerase proteins in the reverse transcriptase family but revealed hallmarks that distinguish them from retroviral and retrotransposon relatives. Thus, the proposed telomerase catalytic subunits are phylogenetically conserved and represent a deep branch in the evolution of reverse transcriptases.

Telomerase is a ribonucleoprotein enzyme responsible in most eukaryotes for the complete replication of chromosome ends, or telomeres (1). Its RNA subunit provides the template for addition of short sequence repeats [typically 6 to 26 nucleotides (nts) to the chromosome 3′ end (2). In ciliated protozoa and yeast, telomerase is regulated and the average telomere length is maintained (3). In most human somatic cells, however, telomerase activity cannot be detected and telomeres shorten with successive cell divisions (4). Telomerase activity reappears in immortalized cell lines and in about 85% of human tumors, which has led to studies of the usefulness of telomerase for cancer diagnostics and therapeutics (5, 6).

Telomerase RNA subunits have been identified and analyzed in ciliates, yeast, and mammals (2, 7), but the protein subunits have been elusive. In Tetrahymena, two telomerase-associated proteins (p80, p95) have been described (8), and p80 homologs have been found in humans and rodents (9); the presence of catalytic active site residues in these proteins has not been established. Purification of telomerase from the ciliateEuplotes aediculatus yielded two proteins, p123 and p43 (10), that appear unrelated to p80 and p95 (11). p123 contains reverse transcriptase (RT) motifs and is homologous to yeast Est2 (Ever shorter telomeres) protein (11), which is essential for telomere maintenance in vivo (12). The RT motifs of Est2p are essential for telomeric DNA synthesis in vivo and in vitro (11, 13), supporting the conclusion that Est2p and p123 are the catalytic subunits of telomerase. The question remained whether there are two telomerases in biology, one based on p80- and p95-like proteins and one on p123/Est2p-like proteins.

To determine if Est2p/p123 is conserved among eukaryotes, we searched for homologs in the fission yeast S. pombe and humans. Polymerase chain reaction (PCR) amplification of S. pombeDNA was carried out with degenerate-sequence primers designed from theEuplotes p123 RT motifs B′ and C. Of the four prominent products generated, the ∼120–base pair (bp) band encoded a peptide sequence homologous to p123 and Est2p. Using this PCR product as a probe for colony hybridization, we identified two overlapping clones from a genomic library and three from a cDNA library (14). None of the three cDNA clones was full length, so we used RT-PCR to obtain the NH2-terminal sequences (15). This putative telomerase reverse transcriptase gene,trt1 +, encoded a basic protein with a predicted molecular mass of 116 kilodaltons (kD) (Fig.1A). The sequence similarity to p123 and Est2p was especially high in the seven RT motifs (Table1) and in motif T (Telomerase-specific) (Fig. 2). Fifteen introns, ranging from 36 to 71 bp, interrupted the coding sequence. All had consensus splice and branch site sequences (16).

Figure 1

The gene for the S. pombetelomerase protein and phenotypes associated with its deletion. (A) The trt1 + locus, the location of the ∼120 bp PCR product that led to its identification, and the regions replaced by ura4 + orhis3 + genes in thetrt1 mutants (K, Kpn I; Xb, Xba I; H, Hind III; Xc, Xca I; Xh, Xho I). (B) Telomere shortening phenotype of trt1 mutants. A  trt1+/trt1 diploid (28) was sporulated and the resulting tetrads were dissected and germinated on a YES (Yeast Extract medium Supplemented with amino acids) plate (29). Colonies derived from each spore were grown at 32°C for 3 days, and streaked successively to fresh YES plates every 3 days. A colony from each round was placed in 6 ml of YES liquid culture at 32°C and grown to stationary phase, and genomic DNA was prepared. After digestion with Apa I, DNA was subjected to electrophoresis on a 2.3% agarose gel, stained with ethidium bromide to confirm approximately equal loading in each lane, transferred to a nylon membrane, and hybridized to a telomeric DNA probe. The Apa I site is located 30 to 40 bp away from telomeric repeat sequences in chromosomes I and II. (C) Colony morphology of trt1 + andtrt1 cells. Cells plated on MM [Minimal Medium (29) with glutamic acid substituted for NH4Cl] were grown for 2 days at 32°C prior to photography. (D) Micrographs oftrt1 + andtrt1 cells grown as in (C).

Figure 2

Structure and RT sequence motifs of telomerase proteins. (A) Locations of telomerase-specific motif T and conserved RT motifs 1, 2, and A through E (24) are indicated by colored boxes. The open rectangle labeled HIV-1 (Human Immunodeficiency Virus) RT delineates the portion of this protein shown in (B). pI, isoelectric point. (B) The crystal structure of the p66 subunit of HIV-1 RT (Brookhaven code 1HNV). Color-coding of RT motifs matches that in (A). The view is from the back of the right hand, which allows all motifs to be seen. (C) Multiple sequence alignment of telomerase RTs and members of other RT families (Sc_al, cytochrome oxidase group II intron 1-encoded protein from S. cerevisiae mitochondria; Dm_TART, reverse transcriptase from Drosophila melanogaster TART non-LTR retrotransposable element). Boldface residues indicate identity of at least three telomerase sequences in the alignment. Colored residues are highly conserved in all RTs and shown as space-filled residues in (B). The number of amino acids between adjacent motifs or to the end of the polypeptide is indicated. TRT con and RT con, consensus sequences for telomerase RTs (this work) and non-telomerase RTs (24) (amino acids are designated h, hydrophobic, A, L, I, V, P, F, W, M; p, polar, G, S, T, Y, C, N, Q; c, charged, D, E, H, K, R). Red arrowheads show some of the systematic differences between telomerase proteins and other RTs. Red rectangle below motif E highlights the primer grip region discussed in the text. 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 S. pombe trt1 + gene and the human TRT cDNA (pGRN121) have been deposited in GenBank (accession nos. AF015783 and AF015950, respectively).

Table 1

Amino acid sequence identity between telomerase reverse transcriptases. Each value is % identity (% similarity in parentheses) based on RT motifs 1, 2, and A through E in Fig 2C.

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If trt1 + encodes the telomerase catalytic subunit in S. pombe, deletion of the gene would be expected to result in telomere shortening and perhaps cellular senescence as seen with the est2 mutants in S. cerevisiae(11, 13). To test this, we created two deletion constructs (Fig. 1A), one removing motifs B′ through E in the RT domain, and the second deleting 99% of the open reading frame (ORF). Haploid cells grown from both types of spores showed progressive telomere shortening to the point where hybridization to telomeric repeats became almost undetectable (Fig. 1B). Senescence was indicated by (i) reduced ability of the cells to grow on agar, typically by the fourth streak-out after germination; (ii) the appearance of colonies with increasingly ragged edges (Fig. 1C); and (iii) the increasing fraction of elongated cells (Fig. 1D). When individual enlarged cells were separated on the dissecting microscope, the majority underwent no further division. The same trt1 cell population always contained normal-size cells that continued to divide but frequently produced nondividing progeny. The telomerase-negative survivors may use a recombinational mode of telomere maintenance as documented in budding yeast strains with deletions of telomere-replication genes (12,17).

A candidate human p123/Est2p/Trt1p homolog was identified by a BLAST search of the EST (expressed sequence tag) database (GenBank AA281296). This EST was the top-ranked match in sequence searches withEuplotes p123 (P = 3.3 × 10−6) and S. pombe Trt1p (P = 9.7 × 10−7). The human EST was not found in searches with yeast Est2p, but subsequent pairwise comparison of these sequences showed a convincing match. Sequencing of the rest of the cDNA clone containing the EST revealed all eight TRT (Telomerase Reverse Transcriptase) motifs, but not in a single ORF (18). We used the sequence information from this incomplete cDNA clone to isolate an extended cDNA clone from a library of 293 cells, an adenovirus E1-transformed human embryonic kidney cell line (19). This cDNA clone (pGRN121) had a 182-bp insert relative to the EST clone, which increased the spacing between motifs A and B′ (18) and put all seven RT motifs and the telomerase-specific motif T motifs in a single contiguous ORF (Fig. 2). RT-PCR amplification of RNA from 293 cells and from testis each gave two products differing by 182 bp (20). The larger and smaller products from testis RNA were sequenced and found to correspond exactly to pGRN121 and the EST cDNA, respectively.

The relative abundance of hTRT mRNA was assessed in six telomerase-negative mortal cell strains and six telomerase-positive immortal cell lines (21) (Fig.3). The steady-state level of hTRT mRNA was higher in immortal cell lines with active telomerase (6) than in any of the telomerase-negative cell strains tested. Telomerase activity was more strongly correlated with the abundance of hTRT mRNA than with that of telomerase RNA (hTR) (7). In contrast, the abundance of mRNA for the human p80 homolog TP1 (9) did not correlate with telomerase activity (Fig. 3). Thus, while our proposal that hTRT is the catalytic subunit of human telomerase is based mainly on protein structural features (similarity of RT motifs, the T motif, molecular mass > 100 kD, pI > 10), the correlation of its mRNA expression level with activity also supports this conclusion.

Figure 3

Expression of hTRT in telomerase-negative mortal cell strains (lanes 1 to 6) and telomerase-positive immortal cell lines (lanes 7 to 12). RT-PCR (21) for hTRT, hTR (human telomerase RNA component), TP1 (telomerase-associated protein related to Tetrahymena p80), and GAPDH (to normalize for equal amounts of RNA template) was carried out on RNA from: (1) human fetal lung fibroblasts GFL, (2) human fetal skin fibroblasts GFS, (3) adult prostate stromal fibroblasts 31YO, (4) human fetal knee synovial fibroblasts HSF, (5) neonatal foreskin fibroblasts BJ, (6) human fetal lung fibroblasts IMR90, (7) melanoma LOX IMVI (8) leukemia U251, (9) NCI H23 lung carcinoma, (10) colon adenocarcinoma SW620, (11) breast tumor MCF7, and (12) human 293 cells.

Sequence alignment of the four telomerase genes revealed features similar to other reverse transcriptases, as well as differences that serve as hallmarks of the telomerase subgroup. The new T motif is one telomerase-specific feature not found in the other RTs examined. Another is the distance between motifs A and B′, which is longer in the TRTs than in other RTs (Fig. 2A). These amino acids can be accommodated as an insertion within the “fingers” region of the structure that resembles a right hand (22, 23) (Fig. 2B). Within the motifs, there are a number of substitutions of amino acids (red arrowheads in Fig. 2C) that are highly conserved among the other RTs. For example, in motif C the two aspartic acid residues (DD) that coordinate active site metal ions (22) occur in the context hxDD(F/Y) in the telomerase RTs compared to (F/Y)xDDh in the other RTs (24). Another systematic change characteristic of the telomerase subgroup occurs in motif E, where WxGxSx appears to be the consensus among the telomerase proteins, whereas hLGxxh is characteristic of other RTs (24). This motif E is called the “primer grip” (23), and mutations in this region affect RNA priming but not DNA priming (25). Because telomerase uses a DNA primer, the chromosome 3′ end, it is not unexpected that it should differ from other RTs in this region. Given that the simple change from Mg2+ to Mn2+ allows HIV RT to copy a small region of a template in a repetitive manner (26), it is tempting to speculate that some of the distinguishing amino acids in the TRTs may cause telomerase to catalyze repetitive copying of the template sequence of its tightly bound RNA subunit.

Using the seven RT domains (Fig. 2C) defined by Xiong and Eickbush (24), we constructed a phylogenetic tree that includes the four telomerase RTs (Fig. 4). The TRTs appear to be more closely related to RTs associated with msDNA (multicopy single-stranded DNA), group II introns, and non-LTR (Long Terminal Repeat) retrotransposons than to the LTR-retrotransposon and viral RTs. The relationship of the telomerase RTs to the non-LTR branch of retroelements is intriguing, given that the latter elements have replaced telomerase for telomere maintenance in Drosophila(27). However, the most striking finding is that the TRTs form a discrete subgroup, about as closely related to the RNA-dependent RNA polymerases of plus-stranded RNA viruses such as poliovirus as to retroviral RTs. In view of the fact that the four telomerase genes are from evolutionarily distant organisms—protozoan, fungi, and mammals—this separate grouping cannot be explained by lack of phylogenetic diversity in the data set. Instead, this deep branching suggests that the telomerase RTs are an ancient group, perhaps originating with the first eukaryote.

Figure 4

A possible phylogenetic tree of telomerases and retroelements rooted with RNA-dependent RNA polymerases. After sequence alignment of motifs 1, 2, and A through E (178 positions, Fig. 2C) from four TRTs, 67 RTs, and three RNA polymerases, the tree was constructed using the Neighbor Joining method (30). Elements from the same class that are located on the same branch of the tree are simplified as a box. The length of each box corresponds to the most divergent element within that box.

The primary sequence of hTRT and eventual reconstitution of active human telomerase may be used to discover telomerase inhibitors, which in turn will permit additional testing of the anti-tumor effects of telomerase inhibition. The correlation between hTRT mRNA levels and human telomerase activity shown here indicates that hTRT also has promise for cancer diagnosis. With an essential protein component of telomerase now in hand, the stage is set for more detailed investigation of fundamental and applied aspects of this ribonucleoprotein enzyme.

  • * Present address: Swiss Institute for Experimental Cancer Research, 1066 Epalinges/VD, Switzerland.

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