- Research article
- Open Access
Insights into the evolution of mammalian telomerase: Platypus TERT shares similarities with genes of birds and other reptiles and localizes on sex chromosomes
BMC Genomics volume 13, Article number: 216 (2012)
The TERT gene encodes the catalytic subunit of the telomerase complex and is responsible for maintaining telomere length. Vertebrate telomerase has been studied in eutherian mammals, fish, and the chicken, but less attention has been paid to other vertebrates. The platypus occupies an important evolutionary position, providing unique insight into the evolution of mammalian genes. We report the cloning of a platypus TERT (OanTERT) ortholog, and provide a comparison with genes of other vertebrates.
The OanTERT encodes a protein with a high sequence similarity to marsupial TERT and avian TERT. Like the TERT of sauropsids and marsupials, as well as that of sharks and echinoderms, OanTERT contains extended variable linkers in the N-terminal region suggesting that they were present already in basal vertebrates and lost independently in ray-finned fish and eutherian mammals. Several alternatively spliced OanTERT variants structurally similar to avian TERT variants were identified. Telomerase activity is expressed in all platypus tissues like that of cold-blooded animals and murine rodents. OanTERT was localized on pseudoautosomal regions of sex chromosomes X3/Y2, expanding the homology between human chromosome 5 and platypus sex chromosomes. Synteny analysis suggests that TERT co-localized with sex-linked genes in the last common mammalian ancestor. Interestingly, female platypuses express higher levels of telomerase in heart and liver tissues than do males.
OanTERT shares many features with TERT of the reptilian outgroup, suggesting that OanTERT represents the ancestral mammalian TERT. Features specific to TERT of eutherian mammals have, therefore, evolved more recently after the divergence of monotremes.
Telomeres are specialized DNA-protein structures at the end of linear chromosomes . Telomeres are important for protecting chromosomes from recombination, and fusion, and also play a role in cellular signaling following DNA damage. As a result of the inability of DNA polymerases to replicate chromosomal ends, telomeres shorten with each subsequent cell division. This progressive telomere shortening ultimately leads to cell growth arrest and senescence . The reduction in telomere length is compensated for by the enzyme telomerase, which is composed of a catalytic subunit (TERT) with reverse transcriptase activity and an RNA template (TR) [3, 4]. In addition to this canonical function, telomerase stimulates cell proliferation, protects against oxidative damage and apoptosis, and modulates gene expression (for review see [5–7]).
Telomerase plays a critical role in aging and cancer in vertebrates [8, 9]. In normal somatic cells telomerase is downregulated, and only cells with high proliferation rates such as male germ line cells, stem cells and cells of the immune system retain high levels of telomerase activity . Most human cancer cells express elevated levels of telomerase which is critical for tumor development . The expression and activity of telomerase is tightly regulated principally at the level of TERT transcription (for review see ). Numerous TERT alternatively spliced (AS) variants have been identified in both vertebrates and plants . The expression of human AS variants is regulated during development and carcinogenesis [14–17]. With the exception of the human AS TERT variant α, which is proposed to be a dominant-negative inhibitor of telomerase activity, the function of the TERT AS variants remains to be determined [18, 19].
TERT genes were initially cloned from Euplotes aediculatus and Saccharomyces cerevisiae[20, 21]. Other TERT genes from yeast – Schizosaccharomyces pombe and Candida albicans[22, 23], and protozoa – Giardia lamblia, Oxytricha, Tetrahymena, Paramecium, Leishmania and Plasmodium have been subsequently described [24–29]. Several TERT genes have been cloned from plants – Oryza sativa, Asparagales, and Arabidopsis[30–33]. The protostomian TERT genes have been identified in a number of insect species including Apis mellifera, Bombyx mori and Tribolium castaneum, and the nematode Caenorhabditis elegans[34–36]. Recently, the TERT gene was cloned from an echinoderm, the purple sea urchin (Strongylocentrotus purpuratus), and urochordates, the sea squirt species Ciona intestinalis and C. savignyi[37, 38]. Orthologs of TERT have been identified in many vertebrates including human, dog, mouse, rat, hamster, chicken, frog (Xenopus laevis), and several fish (Danio rerio, Takifugu rubripes, Nothobranchius furzeri, Oryzias latipes, O. melastigma, and Epinephelus coioides) [22, 39–48]. The TERT gene, however, has not been characterized in the basal group of mammals, the egg-laying monotremes (platypuses and echidnas).
The platypus, Ornithorhynchus anatinus, is an important species for evolutionary studies which possesses a unique combination of mammalian and reptilian features (for reviews see [49–51]). Platypuses lay eggs, but produce milk from mammary glands. Their average body temperature is lower (about 32°C) than other mammals. Many anatomical features of their reproductive system resemble birds including sperm shape and ovarian structure. The platypus genome project has also revealed many similarities between monotreme and avian genomes including genome size and repeat content . Interestingly, monotreme sex chromosomes share high number of orthologues with chicken sex chromosomes but not with the eutherian X chromosome further supporting a special evolutionary position of this species [53–55]. To investigate the evolution of vertebrate TERT we cloned and then characterized the platypus ortholog.
The platypus occupies an important position in the evolution of the synapsid branch of amniotes providing unique insight into the evolution of mammalian genes. In this work we report the cloning of a platypus TERT ortholog, and provide an evolutionary comparison with TERT proteins of other vertebrates. We identified alternatively spliced forms and determined the expression pattern of platypus TERT (OanTERT) mRNA and telomerase activity in various platypus tissues. The chromosomal localization of the OanTERT gene and length of telomeres in the platypus were also defined. Telomerase activity in heart and liver in platypus females and males was compared. All these analyses are presented in the context of the evolution of the vertebrate TERT gene and its role in the regulation of telomerase activity.
Cloning and characterization of platypus TERT cDNA sequences
Telomere repeats were detected at the termini of platypus chromosomes by fluorescence in situ hybridization (FISH) suggesting that the platypus encodes a functional TERT gene [53, 56]. The platypus genome project identified an OanTERT locus . However, in the current NCBI database (as of August 29, 2011) this locus [GenBank:LOC100074692] is classified as a pseudogene due to a number of frameshifts and insertions of repetitive mobile elements in the regions corresponding to exon 1 and 2. Therefore, we cloned the platypus TERT cDNA by RT-PCR using ovarian and brain RNA of an adult platypus and primers based on the selected sequences of the genomic contig containing LOC100074692 [GenBank:NW_001794359.1] (Additional file 1 and Additional file 2: Table S1c and Figure S1a). The 5′ half of the cDNA does not correspond exactly to the released genome sequence due to a misalignment in the 3′ region of the predicted first exon and major gaps in the sequence corresponding to the predicted second exon (Additional file 2: Figure S1b). The positions of the gaps and misalignments correspond to regions of a very GC-rich DNA sequence of low complexity, suggesting that the assembly of the OanTERT locus contains errors due to the complicated secondary structure of the TERT gene as well as the high levels of repetitive sequences found in the platypus genome . The alignment of the cDNA sequence with the genome sequence suggest that the platypus TERT is encoded by 16 exons similar to other vertebrate TERT genes .
The assembled cDNA of OanTERT contains the 4522 nucleotide (nt) sequence with 2 nt of the 5′ UTR, a reading frame of 3891 nt including start and stop codons, and 629 nt of the 3′ UTR (Additional file 2: Figures S1c, S1d, S1e). The UTR sequences are not complete. If the closest of two possible polyadenylation signal sequences (AATAGA, AAGAAA) located in genomic DNA are used to terminate transcription, then the size of platypus 3′ UTR would be 700–800 bp, in the size range of the 3′ UTRs found in TERT genes of other vertebrates. The open reading frame (ORF) encodes an Arg/Leu-rich protein of 1296 amino acids with a predicted molecular weight of 146 kDa. The successful cloning of OanTERT cDNA indicates that platypus expresses an mRNA with a continuous reading frame that encodes a functional TERT protein.
The position of platypus TERT in the evolution of vertebrate TERT genes
To evaluate the evolutionary relationship of OanTERT to other TERT proteins we have aligned its amino acid sequence with available TERT sequences of different metazoan species. Most of the TERT sequences (20 sequences) were derived from annotated GenBank files. Five TERT sequences, those of chimpanzee (Ptr), elephant (Laf), opossum (Mdo), zebra finch (Tgu), and anole (Aga) were molecular models from GenBank or Ensembl and these were subjected to small scale corrections as described in Methods and Additional file 3. Four TERT sequences, those of wallaby (Meu), elephant shark (Cmi), leech (Hro), and hydra (Hma) were assembled de novo using the genomic data from GenBank (Methods and Additional file 3).
Initial evaluation was focused on the evolutionary conservation of the functional domains in OanTERT and included full-length TERT sequences of platypus and seven species representing different vertebrate systematic groups (Figure 1a). The analysis included platypus (Oan), human (Hsa), mouse (Mmu), gray short-tailed opossum (Mdo), domestic chicken (Gga), green anole lizard (Aca), African clawed toad (Xle), and pufferfish (Tru) proteins. Conserved domains and motifs detected in the aligned sequences are shown in Figure 1a (see also Additional file 4: Figure S2). Platypus TERT contains all four essential domains present in vertebrate TERT proteins: TEN (telomerase essential N-terminal), TRBD (telomerase RNA-binding domain), RT (reverse transcriptase), and CT (C-terminal) . Inside these domains all of the conserved motifs were found, including the GQ motif in the TEN domain, motifs v-II, v-III, QFP, and T in the TRBD, and motifs 1, 2, 3, A, IFD, B′, C, D, and E in the RT domain. In many of these conserved domains as well as in the entire molecule the platypus TERT protein has the highest sequence similarity to the marsupial (opossum) TERT with the next highest similarity with birds and the lizard, followed by eutherian mammals (human, mouse) and the amphibian (toad) (Figure 1b).
The main differences in TERT primary structure among the vertebrate species are in the length and the sequence of the three linker regions: linker L1 between TEN and TRBD, and linkers L2 and L3 inside the TRBD, between motifs v-II and v-III, and v-III and QFP, respectively (Additional file 4: Figure S2). Analysis of TERT protein sequences of 28 metazoan species revealed that they fall into three different groups based on the size of linkers (Figure 1c). Those with long linker elements have cumulative linker size equal or longer than platypus and include marsupials, sauropsids, a shark (Cmi) and an invertebrate - sea urchin (Spu). Short TERT proteins have cumulative linker size shorter than 60% of the size of these regions in platypus TERT. These proteins are found in all eutherian mammals and ray-finned fish and some invertebrates. Only two species have linker size intermediate between these two extremes - an amphibian (Xle) and an invertebrate (Hro). The shorter TERT proteins also often contain small reductions in the size of the more conserved parts of the protein, especially in the beginning of the RT domain 3 (Additional file 4: Figure S2).
To further refine the evolutionary position of the OanTERT gene we have constructed phylogenetic tree of vertebrate TERT sequences (Figure 1d). The tree is based on the alignment of the longest available sequence for each protein. Setting gap tolerance to 0%, we have removed all aligned columns that contained gaps. These gaps were results either of low conservation or incompleteness of sequences. The tree confirmed that monotreme TERT is most closely related to the marsupial TERT. The monotreme and marsupial TERT proteins assume an intermediate position between TERTs of sauropsids (including birds) and the TERTs of eutherian mammals indicating that they share sequence similarities with the proteins from both of these groups. Interestingly, both monotreme and marsupial TERTs are positioned closer to the TERT of the common ancestor of sauropsids than that of the ancestor of eutherian mammals. The tree also indicates a close relationship of all vertebrate TERT proteins with long linkers as these are concentrated in one part of the tree close to base of vertebrate tree. Because the tree is constructed mostly from the sequence of the conserved regions, this analysis suggests that the relationship of the vertebrate proteins with long linkers is also paralleled by the sequence similarities in conserved domains.
In conclusion, monotreme and marsupial TERT proteins are evolutionary closely related to sauropsid TERT.
Identification of alternatively spliced TERT variants
Seven alternatively spliced events were identified in platypus TERT that yield seven single AS forms (Figure 2a, see also Additional file 2: Figure S1a). Four of them (A, A2, A3, and A4) delete part of exon 2. The AS forms A and A3 use a novel splice donor (SD) located in the first quarter of exon 2. The AS variant A uses a splice acceptor (SA) site of the second intron and the AS variant A3 employs the SA of the third intron. Similarly, the AS variants A2 and A4 use a novel SD site located in the last third of the second exon in combination with a SA in the second intron (A2) or the third intron (A4). The additional AS variant, variant B, deletes the third exon and the sixth AS variant, variant C, has a deletion of the seventh, eighth and the part of the ninth exon. Finally, variant D was identified in a single clone from brain cDNA and contains an insertion cassette of 25 nucleotides derived from the first intron. The AS variants A and A4 retain ORF and AS variants A3, A2, B, C, and D contain premature termination codons (PTCs). The splicing events A to A4, and B are mutually exclusive, but each of them may be combined with C or D in one AS molecule.
The comparison of platypus AS forms with chicken and human isoforms revealed some similarities. AS forms involving the second exon have been described in human and chicken TERT (, unpublished data, and Figure 2). The chicken AS variant A is structurally similar to the platypus variant A. This form uses a novel SD site in second exon, located in close proximity to the platypus SD site in combination with a SA of the second intron and retains an ORF. Like the platypus A3, the chicken A3 variant containing a PTC uses this novel SD site in combination with a SA site of the third intron. In contrast, all human AS variants involving the second exon contain PTCs and only one of them uses a novel SD site while the three others employ a SD of the first intron. An additional similarity between chicken and platypus alternative splicing was revealed by the identification of the platypus AS variant B, which is identical to the chicken variant V3. The platypus variant C (Δ7-8, and part of 9 exon) has no precise analog among chicken or human AS variants, but chicken variant G (Δ8) and human AS variant β (Δ7-8) delete a similar region and all these forms contain PTCs. These results suggest that, although only seven AS variants of OanTERT have been identified, they have strong structural similarity to several chicken AS variants including AS form A which retains an ORF.
The low conservation of the second exon correlates with the evolution of novel splice sites
The second exon is the least conserved of all vertebrate TERT exons (Figure 1a). The conserved regions are at the beginning and the end of this exon that encode parts of the TEN and TRBD, respectively. These domains are separated by a linker with high sequence variability. In birds and mammals at least six novel SD and one SA sites evolved within the second exon (Figure 2b). In the sequence encoding the TEN domain there are two SD sites that create AS forms which contain PTCs. The first site (hSD) is involved in splicing of the human AS Δ2p(136-end) variant and the SD site (chSD, qSD, mSD) was identified in chicken and quail variant A2, as well as mouse AS TERT variants. Additional new SD and SA sites were identified in the second exon downstream from the sequence encoding the TEN domain. Interestingly, all novel SD sites involved in the production of the AS TERT variants that retain an original ORF are located downstream from the TEN domain. The two SD sites used by platypus and chicken AS TERT variants A (OanSD A/A3, chSD A/A3) are located in a sequence encoding the first hundred amino acids downstream from this domain. The SD site used by the platypus in-frame AS variant A4 (OanSD A2/A4) is also downstream from the TEN domain. This analysis suggests that the second exon, which has high sequence variability, was used to create the novel SD and SA sites during evolution. As a rule, all the AS TERT variants which retain the original ORF are downstream from the TEN domain, and potentially encode variants of TERT with the DNA-binding domain intact.
The expression of alternatively spliced TERT variants in platypus tissues
The expression of TERT mRNA in a variety of platypus tissues was determined by RT-PCR using a set of eight specific primers (Figure 3a). Initially we determined the level of total TERT transcripts using primers that amplified sequences located between exons 14 and 16 which are not known to be involved in alternative splicing (primers 1/2, Figure 3b). Platypus TERT was expressed in all tissues tested: spleen, intestine, liver, kidney, lung, ovary, testes, brain, heart, and skeletal muscle. Surprisingly, the highest TERT mRNA expression was present in brain and skeletal muscle, the organs in which the expression of TERT is very low or absent in most of homeotherms .
The relative levels of the AS variants (with exception of rarely expressed variant D) in the various tissues was also determined (Figure 3c). First, the forward primers specific to different sequences in the second exon and the reverse primer specific to a sequence located in the fifth exon were employed (3/6, 4/6, 5/6). The expression of variant A with an original ORF was detected in liver, lung, ovary, and testes and the expression of the smaller A3 variant, a PTC-containing variant, in intestine, lung, ovary, testes, brain, and skeletal muscle. The A2 variant, containing a PTC, was detected in all tissues except lung and skeletal muscle. In contrast, the A4 variant with an original ORF was detected only in ovary, testes, and skeletal muscle. If the forward primer located within the sequence that is deleted in all the A variants (primer 5) was used in combination with reverse primer 6, then the OanTERT was detected in all tissues. The products of this reaction represent part of wild-type (WT) TERT in combination with AS variant B in different relative ratios. Finally, additional PCR determined that the variant C was expressed in two tissues, in brain and testes (primers 7/8).
In summary, this analysis revealed that all variants were detected only in the testes (Figure 3d). The ovary expressed most variants except the rare variant C. Other tissues expressed different combinations of three to four AS TERT variants, suggesting that the alternative splicing of TERT is regulated during cell differentiation. In conclusion, the expression of TERT mRNA is high in most platypus tissues. Although, a significant number of TERT mRNAs are alternatively spliced, WT TERT transcripts were detected in all tissues suggesting that telomerase activity is present in these tissues.
Telomerase activity is ubiquitous in platypus tissues
To investigate whether telomerase is active in most platypus organs as suggested by the TERT mRNA expression analysis, tissue extracts prepared from organs of an adult male platypus were analyzed by a telomerase repeat amplification protocol (TRAP) assay (Figure 3e, lanes 1–9). Telomerase activity was detected in all evaluated tissues, although at different levels. The highest levels of telomerase activity were detected in testes and intestine, following by spleen, lung, liver, skeletal muscle, and brain. The lowest levels of telomerase activity were detected in kidney and heart. In most homeotherms telomerase activity is very low or undetectable in heart, skeletal muscle, and brain [60–62]. Therefore, we determined telomerase activity in these tissues obtained from three different adult female platypuses and directly compared it to telomerase activity in these tissues obtained from female mice and chicken (lanes 11–31). Telomerase activity was detected in heart, skeletal muscle, and brain in all three platypuses, though variations among individuals were observed. Similar levels of telomerase activity were detected in mouse tissues (lanes 20–28). Individual variability was also present despite that two of the three females were from the same litter and all three animals were of the same age. In contrast, chicken heart and muscle did not expressed telomerase activity and very low levels were detected in chicken brain (lanes 29–31). These results show that telomerase activity is present in all nine analyzed platypus tissues. As in laboratory mice, telomerase activity was expressed also in tissues with low cell proliferation.
Platypus telomere length
The average length of telomeres in mammals ranges from very long (up to 50 kb in some rodents and lagomorphs) to very short (below 10 kb in primates). However, species can be divided into two groups according to their telomere length, one group with the average telomere length below and the second above 20 kb . Length of platypus telomeres was determined by telomere restriction fragment (TRF) analysis and their size was compared to the telomere length of other vertebrates (quail, duck, chicken, mouse, human, toad, and opossum) (Figure 4a). Platypus telomeres were above 20 kb (approximately 30–40 kb long) similarly to quail, chicken, mouse, toad and opossum. In contrast, human and duck had much shorter telomeres (4–10 kb). In conclusion, platypus belongs to the group of animals with long telomeres.
In terms of physical distribution of telomere repeats, frequent interstitial telomere sequences (ITS) have been reported in birds, frogs, and marsupials [64–67]. The ITS (discontinuous bands of molecular weights below 10 kb) were visible in all birds, toad and platypus, but they were undetectable or absent in human and mouse tissues. The FISH analysis confirmed the presence of end-terminal telomeric repeats and revealed an extensive ITS on chromosome 1 in platypus genome (Figure 4b). The strong ITS could possibly interfere with the detection of telomeric sequences at the end of chromosomes. However, the strength of this signal is much greater than signals at the ends of chromosomes on the regular FISH analyses so if this sequence is formed from uninterrupted telomeric repeats then it would not appear on the gel because its high molecular weight. We rather expect that this ITS is interspersed by nontelomeric sequences containing sites for frequently cutting restriction enzymes and appear in the gel as fragments below 4 kb like the ITS of birds .
Chromosomal localization of platypus TERT
The platypus physical map is relatively sparse and only 15% of known genes have been mapped to chromosomes . Additional mapping primarily concentrated on the gene content of the platypus sex chromosomes and platypus orthologs of the human X chromosomal genes [54, 55, 69]. While the location of the TERT locus-containing contig has not been established in these mapping projects, preliminary synteny analysis of TERT-containing chromosomes (human chromosome 5 and chicken chromosome 2) with platypus chromosomes suggested the possibility that OanTERT may map to the platypus sex chromosomes. In fact, initial FISH hybridization employing the BAC clone 606P3 localized OanTERT on a pair of small metacentric chromosomes of differing sizes (Figure 5a). Co-hybridization with BAC clone 462c1, which contains the major histocompatibility complex (MHC) class I and class II genes that were previously localized to X3q/Y3 , established that these chromosomes are X3 and Y2 and that OanTERT is located in the pseudoautosomal region of X3p/Y2q (Figure 5 b,c).
The physical mapping of OanTERT contig genes extended previously described homology between human chromosome 5, chicken chromosome 2 and platypus X2/Y2/X3 [54, 55]. Detailed synteny analysis of the genes surrounding platypus and human TERT shows that the entire section of human chromosome 5 that shares synteny with chicken chromosome 2 (the terminal region of 31.5 MB shown in Additional file 6: Table S2) contains 70 protein-coding genes. Orthologs of 61 of these genes were found in the platypus genome and 90% of these are now mapped to chromosomes. Almost all of the mapped genes are located on the short arms of platypus sex chromosomes X2 (7 genes) and X3 (46 genes). Only one gene (SRD5A1 ortholog) is located elsewhere, on sex chromosome X1.
The short arms of platypus X2 and X3 chromosomes that contain orthologs of human genes located at human chromosome 5 (listed in Table S2, Additional file 6) also contain orthologs of human genes located on human chromosomes 3, 4, 6, 9, 17, and 18 (Figure 5d). A majority of these genes (as well as 5p-orthologs) are part of one ancient linkage group conserved on chicken chromosome 2 (approximately between 55 and 95 MB). Only a few X2p/X3p-genes do not belong to this group, including the orthologs of MLLT3 PTPLAD2 group, CISH, and NAF1. One additional exception, identified in this work, is a group of six genes with human orthologs located at 17p13 (sharing synteny with chicken genes localized on chromosome 19). Incorporation of these genes into a linkage with genes which orthologs map to chicken chromosome 2 appears to be monotreme-specific.
Sex differences in telomerase expression
The localization of the OanTERT gene on sex chromosomes and difference in telomerase activity between male and female heart tissues (Figure 3e) prompted us to analyze telomerase activity in male and female platypus tissues. Telomerase expression in liver and heart was analyzed since differences in telomerase expression or telomere length in these tissues have been described in the rat [70, 71]. Material was obtained from adult platypuses caught from actively breeding wild populations, but their precise age is not known. However, results presented in Figure 3e demonstrated that individual variability in telomerase activity in these platypuses is not greater than individual variability in inbred mice at adulthood. We determined the levels of telomerase activity in tissues from three male and female animals (Figure 6). Despite variations between individuals, the results clearly demonstrated that on average females expressed higher levels of telomerase activity in both tissues than males.
Platypus TERT and its position in the evolution of vertebrate TERT genes
Analysis of the platypus TERT gene creates an important framework for the evolutionary comparison of TERT genes of eutherian mammals and sauropsid reptiles (including birds). Platypus, one of a few surviving species of monotremes, has a unique evolutionary position. Monotremes, followed by marsupials, first split from all other contemporary mammals after the divergence of the synapsid lineage leading to mammals and the sauropsid lineage leading to contemporary reptiles and birds . Monotremes and marsupials, therefore, might retain some features of the pre-mammalian synapsid TERT gene that were lost in eutherian mammals. The higher levels of sequence similarity of four principal TERT domain of OanTERT to the marsupial, and avian TERT than to the eutherian TERT support this hypothesis. Moreover, platypus and marsupials also retain three extended linker regions of a non-conserved sequence in the N-terminal region of the TERT protein which are about twice longer than the linkers in eutherians and their size matches more closely the linkers in sauropsids.
Interestingly, the long and intermediate linker regions present in most of the tetrapods except the eutherian mammals were not found in the well-studied group of ray-finned fish but are present in elephant shark TERT. Elephant shark belongs to the oldest evolutionary group of contemporary jawed vertebrates . It is, therefore, possible that the long TERT of the shark may represent the original jawed-vertebrate TERT structure and that the linkers were shortened independently in ray-finned fish and eutherian mammals. Divergent evolution of fish TERT is supported by the evolutionary tree built mostly on the conserved TERT sequences outside the linker regions that situates the elephant shark TERT closer to the tetrapod TERT and farther from ray-finned fish TERT (Figure 1d). These changes could be, at least partly, a consequence of genome duplication and increase in mutation rate in the teleost branch of ray-finned fish [72–75]. The long TERT structure described in sea urchin suggests an even deeper evolutionary root of the extended linker .
The long-term evolutionary stability of the extended linker regions suggests a possible function that constrains evolutionary change. The invertebrate sea urchin has two TERTs with extremely long linker regions and a specific function in embryogenesis were suggested for these structures . In vertebrate the presence of the longer linker region in TERT proteins of different species correlates with evolutionary distribution of interstitial telomeric sequences (ITS). ITS were detected in the platypus genome and while these repeats were described in most vertebrate species they occur at a higher frequency in frogs and birds but not eutherian mammals [64, 65, 67]. Several mechanisms contribute to the formation of ITS depending on their length and sequence organization [76, 77]. For the formation of most of the short ITS the direct involvement of telomerase during the repair of double-stranded breaks has been implicated. Therefore, one of possible functions of the extended linker region of the TERT protein might be to increase the recruitment of telomerase to sites of double-strand breaks. The precise function of the extended linker of TERT however remains to be established.
The shortening of the once extended TERT molecules could be also restricted by the necessity to adapt to interacting molecules. One of such molecule could be the telomerase RNA (TR) because the three TERT linkers are either inside or close to the TR-binding domain. The minimalist size of the TR that developed in ray-finned fish correlates with an extensive reduction of the linkers in the fish TERT protein . Further, the largest vertebrate TR has been described in sharks and the predicted size of the elephant shark TERT is also the largest of all vertebrates . However, the TERT-TR size correlation does not hold in birds. Birds encode a large TERT protein but the TR is of a similar size as that of mammals .
The evolution of AS variants
Alternative splicing plays an important role in creating proteome diversity in multicellular animals . More than 95% of human intron-containing genes express one or several alternatively spliced variants. While the alternative splicing of TERT transcripts has not been described in protists, it occurs extensively in multicellular organisms such as plants and Metazoa (for recent review see ). All vertebrate TERT transcripts are currently believed to be alternatively spliced, however, there are great differences in frequency and structure of AS variants . The largest number of AS TERT variants have been identified in chicken and human cells, 37 and 21, respectively ([58, 81–85] and unpublished data). In fish, amphibians and rodents only a few variants are known . In contrast to plants, almost all the AS TERT variants differ in structure between mammals and birds. There is only one known example of AS variants which is structurally identical, an in-frame deletion of exon 10 in the chicken and rat [58, 86]. We identified seven AS variants of OanTERT which likely represent all the major platypus TERT AS variants. The variant B is identical to chicken variant V3 and surprisingly, the structure of three other variants (A, A3, C), also resembles the chicken AS isoforms. This unprecedented conservation of AS variants between a basal mammal lineage and a bird suggests an important function for alternative splicing in TERT regulation. It also suggests that the repertoire of AS TERT variants identified in eutherian mammals evolved recently.
Two AS sites evolved in the second exon of OanTERT which yield four AS variants, two of which retain an ORF. In contrast to variants containing PTCs, which are targeted by nonsense mediated decay (NMD), these AS variants will likely produce TERT proteins . The A variant, which maintains an original ORF, is highly expressed in liver, lung, ovary, and testes, like the corresponding chicken variant A. Interestingly, the chicken and platypus A variants, as well as AS variant A4, have maintained the TEN domain which plays a role in DNA-binding, however, based on their structure, they would not possess telomerase activity [88, 89]. Several telomerase activity-independent functions have been attributed to the TERT protein including the enhancement of cell proliferation. The alternatively spliced protein-coding TERT isoforms might provide some of these functions [6, 7].
The tissue-specific expression of TERT
Telomerase activity is downregulated during development in most somatic cells simultaneously with a reduction in their rate of proliferation and subsequent differentiation . This process is accompanied by the transcriptional downregulation of TERT. However, the levels of repression differ between cold-blooded (fish and amphibian) and warm-blooded species (mammals and birds). In adult organs of many mammals and birds, the expression of TERT is repressed, with very low or undetectable levels expressed in heart, skeletal muscle and brain [11, 41, 62, 90]. In contrast, the expression of TERT in all adult organs of fish remains at high levels [40, 42, 48]. Likewise, telomerase activity is widespread in the tissues of the toad Xenopus tropicalis including heart, brain, and muscle . However, there are significant differences even among mammals [63, 92]. Most mammals with telomeres longer than 20 kb, including small rodents, bats, some felines, lagomorphs, elephant shrews, and the Virginia opossum, express significant levels of telomerase in adult somatic cells. The platypus, with telomeres longer than 20 kb and telomerase activity in all adult tissues, belongs to these species. Telomerase activity in the platypus was even detected in skeletal muscle, brain, and heart, though in lower levels than in other organs with higher cell proliferation rates. The pattern of telomerase expression in platypus, as well as the length of its telomeres, correlates well with its relatively small size (1–2 kg), supporting the hypothesis that telomerase activity and telomere length inversely correlate with body mass in mammals .
The location of OanTERT on the X3/Y2 sex chromosomes
The chromosomal position of platypus TERT is, to our knowledge, the first instance among amniote vertebrates to localize TERT on sex chromosomes. The homology of platypus and echidna Y2/X3 chromosomes suggests that this is common in monotremes . TERT genes of the other amniotes analyzed to date are located on autosomes [43, 93]. Platypus sex chromosomes have large regions orthologous to avian ZW chromosomes while they generally lack orthologs of genes located at therian XY sex chromosomes [53–55]. Interestingly, the region on human autosome 5 containing the TERT gene is located in proximity to a region homologous to a part of the avian sex chromosome Z . Further, the inspection of the Ensembl database shows that similar juxtaposition is also found in other mammals, including macaque, marmoset, mouse, cow, pig, horse, and opossum. These observations lead to an interesting possibility that the last common ancestor of all mammals might have the TERT gene chromosomal region associated with an avian-type Z chromosome. The TERT position on sex chromosomes may not be an evolutionary innovation of monotremes, but one of the features of mammalian ancestors preserved in the platypus genome and only lost in therian mammals as a result of the evolution of the new XY sex chromosomes .
Comparison of the genes surrounding the TERT locus in platypus, human, and chicken (Figure 5c and Additional file 6: Table S2) suggests that the formation of a discontinuous sex chromosome chain in platypus lead to the relocation of some genes to different chromosomes. The genes found in one region in human chromosome 5 and chicken chromosome 2 are divided into three X chromosomes (X1, X2, and X3) in platypus. Similarly, chicken Z-homologous genes are located on four platypus X chromosomes (X1, X2, X3, and X5) . This suggests that the evolution of the sex chromosome chain might have created instability and possibly increased the rate of interchromosomal rearrangements of the chromosomes involved [95, 96].
The OanTERT locus is located in a pseudoautosomal region of the platypus sex chromosomes. This location, however, does not automatically guarantee identical regulation of the X and Y copies of TERT. Different spacing of two BAC clones (165 F5, 151 O20) hybridizing spots at Y2q and X3p might indicate different chromatin folding and epigenetic state of these two chromosome arms or alternatively incomplete homology . These differences may lead to differential expression of the two TERT copies.
Gender-dependent telomerase expression
Gender-specific differences between telomere length in several mammalian species are well established. Female mice and rats have significantly longer telomeres than age-matched males [71, 97]. In human, female telomeres are longer than male, with the exception of the Amish people [98–100]. These differences in telomere length positively correlate with life expectancy [101, 102]. Telomere length is the result of a complex regulation involving both the synthesis and attrition of telomeric repeats [103, 104]. Telomerase activity is responsible for telomere synthesis and generally correlates with telomere length. Therefore, telomerase activity likely contributes to gender-specific differences in telomere length and, in turn, life expectancy. However, very limited information is available concerning gender-specific telomerase activity in mammals. Cardiac myocytes obtained from female rats express significantly higher levels of telomerase than male cells . Our results demonstrating that female platypuses express higher telomerase activity than males in at least two tissues, liver and heart, extend this observation. Telomerase activity in these tissues may also correlate with lifespan. While this issue will require further study, there are indications that the lifespan of the female platypus in the wild is greater than that of the male. Studies performed over a 30 year period in the upper Shoalhaven River demonstrated that the recapture rate is significantly biased toward females . In addition, the oldest female captured in these studies was 21 years old, while the oldest male was 7.
Recently, it has been shown that there are gender differences in telomere length in the kakapo, Strigops habroptilus, a parrot endemic to New Zealand . The male kakapos have longer telomeres than females which is in distinct contrast to mammals. Previously, it has been suggested that estrogen in mammals is at least partly responsible for the increased telomere length in females since estrogen is a positive regulator of TERT transcription . Female birds, however, also express high levels of estrogen like their mammalian counterparts, therefore, this mechanism cannot apply to birds [108, 109]. Moreover, in contrast to human, where paternal inheritance of telomere length to daughters and sons have been described, the kakapo telomere length is inherited maternally to sons [110, 111]. To reconciliate these opposing patterns of regulation and inheritance of telomere length it has been proposed that heterogametic sex (either XY in mammals or ZW in birds) chromosomes X and Z have a central role in determination of gender-specific differences in telomere length.
The chromosomal localization of TERT on platypus sex chromosomes together with gender-specific expression supports this hypothesis and suggests that gender-specific differences in telomere length are related to the original localization of the TERT gene on sex chromosomes. One possible scenario is that the TERT activator was located on the sex chromosome of a common ancestor of mammals and birds. This localization might be retained in monotremes with the hypothetical TERT activator located on a X specific part of the X chromosome which in platypus may be only partially dosage-compensated . The large gender-specific differences in telomerase expression as well as the lifespan of platypuses may be the result of concomitant localization of TERT and its activator on sex chromosomes. A large part of the platypus sex chromosomes is orthologous to sex chromosome Z in birds, which also does not involved a chromosome-wide dosage compensation mechanism [54, 113, 114]. The localization of a TERT activator on the avian Z chromosome could result in higher levels of telomerase activity in male than female birds because the expression of genes localized on bird sex chromosomes is also weakly dosage-compensated. Finally, therian mammals developed a new sex chromosome system and evolved additional levels of regulation beyond the already existing regulatory circuits . In case of TERT gender-specific regulation this relationship could remain conserved in therian mammals while subjected to a level of additional control of the new evolved X chromosome.
The characterization of platypus TERT and its comparison with other vertebrate TERT proteins revealed that it shares many features with birds and other reptiles suggesting that it represents the ancestral mammalian TERT. Structural and regulatory features specific to TERT of eutherian mammals have, therefore, evolved more recently after the divergence of monotreme mammals. Additionally, the results suggest possible relationship between the chromosomal localization of TERT and gender-specific expression of telomerase.
The sequences of platypus TERT cDNA and alternatively spliced TERT variants were submitted to GenBank under accession numbers [GenBank:JF441065, GenBank:JF441066, GenBank:JF441067, GenBank:JF441068, GenBank:JF441069, GenBank:JF441070, GenBank:JF441071, GenBank:JF441072]. The platypus TERT cDNA sequence is a composite of overlapping cDNA clones (Additional file 2: Figure S1a). Due to the extensive TERT alternative splicing which occurs in platypus tissues, secondary structures, and limited tissue resources we were not able to isolate a contiguous TERT clone.
Animals, cell lines, and tissue culture
Material used in this study was obtained from adult wild male and female platypuses (The University of Adelaide Animal Ethics Committee permit no. S-49-2006 to F.G.). Quail (Coturnix japonica) embryonated eggs were obtained from University of Texas, Austin. Embryonated duck (Anas platyrhynchos) eggs (Khaki Campbell) were obtained from McMurray hatchery (Webster City, IA). Embryonated eggs from pathogen-free White Leghorn chickens (Gallus gallus) (the SPF-SC strain) were obtained from Charles River SPAFAS (North Franklin, CT). Mice (Mus musculus) (BALB/c, 2 month-old females) were from University of Texas, Austin and opossum tissues (Monodelphis domestica) (9 week-old male) from the University of New Mexico, Albuquerque.
Total RNA was extracted from frozen tissues using TRIzol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer’s instructions.
Isolation of platypus TERT and its AS variants
The platypus TERT clones were RT-PCR amplified from cDNA obtained from platypus brain testes, ovary, heart, and muscle using 15 distinct combinations of primers Table S1c, Additional file 1). The PCR strategy is shown in Figure S1a (Additional file 2). The amplified fragments were cloned into pGEM-T Easy (Promega, Madison, WI) or into pCR2.1-TOPO (Invitrogen) vectors. Forty four clones were isolated and sequenced.
The TERT protein sequences from species other than platypus were obtained from different sources. Most of the TERT sequences were obtained from annotated GenBank files: Homo sapiens [GenBank:NP_937983.2], Macaca mulatta [GenBank:NP_001177896.1], Canis lupus [GenBank:NP_001026800.1], Bos taurus [GenBank:NP_001039707.1], Mus musculus [GenBank:NP_033380.1], Rattus norvegicus [GenBank:NP_445875.1], Mesocricetus auratus [GenBank:AAF17334.1], Gallus gallus [GenBank:NP_001026178.1], Coturnix japonica [GenBank:ABG75863.1], Cairina moschata [GenBank:ABO65149.1], Xenopus laevis [GenBank:NP_001079102.1], Danio rerio [GenBank:NP_001077335.1], Nothobranchius furzeri [GenBank:ACN38321.1], Oryzias latipes [GenBank:NP_001098286.1], Oryzias melastigma [GenBank:ABB92622.1], Epinephelus coioides [GenBank:ABC47309.1], Takifugu rubripes [GenBank:AAX59693.1], Strongylocentrotus purpuratus [GenBank:NP_001165522.1, GenBank:NP_001123288.2], Daphnia pulex [GenBank:EFX76361]. Two TERT sequences from GenBank were predictions by automatic computational analysis (Pan troglodytes [GenBank:XP_001141571.1], Monodelphis domestica [GenBank:XP_001369432.1]). Three model TERT sequences were obtained from the Ensembl database (http://www.ensembl.org), specifically TERT protein sequences of Loxodonta africana [Ensembl:ENSLAFP00000010943], Taeniopygia guttata [Ensembl:ENSTGUP00000008676], and Anolis carolinensis [Ensembl:ENSACAP00000001407]. The two predicted sequences from GenBank and all three protein models from Ensembl were checked for accuracy by alignments to genomic sequences as well as by multiple protein alignment and small scale corrections were made when necessary (for details see the Additional file 3, p. 1–2). Four TERT sequences were assembled de novo from the genomic data available in GenBank. Two complete assembled sequences (Hydra magnipapillata, Helobdella robusta) were submitted to GenBank under accession numbers [GenBank:BK008019, GenBank:BK008020]. The gapped, incomplete sequences (Macropus eugenii, Callorhinchus milii) are provided in the supplement (Additional file 3, p. 3–4). Protein sequence alignments were constructed by the ClustalX program . The alignment of all sequences is shown in Figure S3 (Additional file 5, PDF version) and in Additional file 7 (text version). The boundaries of the protein domains and motifs were determined based on previously described vertebrate, invertebrate and protist TERT proteins [38, 44, 88, 89, 117–120]. The boundaries are shown in the Figure S2 (Additional file 4). The number of similar amino acids was determined using GeneDoc computer program (http://www.psc.edu/biomed/genedoc). Amino acid similarity is calculated based on BLOSUM62 matrix (BLOSUM35 matrix gave very similar results - data not shown). The percentage is shown relative to the number of residues in OanTERT or in OanTERT functional domains.
The evolutionary tree was constructed by Bayesian inference phylogenetic method. First, the protein sequences were aligned by ClustalX (Additional file 5: Figure S3). After deletion of sequences from the alignment that were not used for building of the tree, all aligned columns containing either gap or unidentified amino acid (X symbol) were removed using Gapstreeze tool software by setting the gap tolerance to 0% (Los Alamos HIV Sequence Database; http://www.hiv.lanl.gov/content/sequence/GAPSTREEZE/gap.html). The resulting alignment consisted of 618 columns of aligned amino acids. Bayesian analysis was performed by MrBayes 3.2.1 program . Evolutionary models implemented by the program included equal rate variation across sites and the fixed-rate amino acid substitution model (aamodelpr = fixed(jones)). In preliminary tests, this fixed-rate amino acid substitution model was repeatedly preferred by the program for this set of protein sequences when mixed amino acid model was applied. Two Bayesian analyses each consisting of four Metropolis-coupled Markov chain Monte Carlo were run in parallel for 250,000 generations and sampled every 100th generation. Convergence of both analyses was assessed using a plot of the generations versus the log probability of the data. The consensus tree was created with burn-in value set to 625. The tree was plotted by the tree-drawing program Dendroscope .
The information about platypus, human, and chicken genes used in synteny analysis (Figure 5d and Additional file 6: Table S2) was retrieved from the latest Ensembl database (Ensembl genes 63). In most cases, the orthology of platypus genes with human and chicken genes corresponded to that assigned by the Ensembl database. For two human genes, GABBR2 and PAPD7, we have assigned orthology to platypus genes based on their position within the same contiguous region in both species and based on high similarity of their protein products established by BlastP program.
Tissue specific expression of platypus TERT by RT-PCR
Total RNA from platypus tissues (4 μg) together with 1 μl of random-hexamer primers were denatured for 10 min at 80°C in 11 μl of water. First strand cDNA synthesis was carried out with 15 U of ThermoScript RT (Invitrogen), 2 μl of 10 mM dNTP, 1 μl of RNaseOUT and 2 μl of 100 mM dithiothreitol at 50°C for 1 hour. The reaction was stopped by 5 min incubation at 85°C. The RNA was destroyed by RNase H treatment and the reaction was diluted with 20 μl of water. For detection of TERT, its AS variants, and GAPDH by RT-PCR, 2 μl of the first-strand synthesis reaction was amplified by 2.5 U Herculase Hotstart DNA polymerase (Agilent, Santa Clara, CA) with appropriate primers (Additional file 1: Table S1).
Telomerase Repeat Amplification Protocol (TRAP)
The level of telomerase activity in various tissues was evaluated using the TRAP assay . Whole cell extracts were prepared with CHAPS buffer . Equivalent amounts of protein extracts (20 μg of total protein) were first incubated with 0.1 μg of the unlabeled TS primer and all four dNTPs (50 μM each) in TRAP reaction buffer, 0.8 mM spermidine, and 5 mM β-mercaptoethanol in a total reaction volume of 50 μl for 45 min at 37°C. All oligonucleotides - TS, ACX, NT, TSNT were described previously . The reaction was stopped by incubation at 94°C for 2 min. Aliquots of synthesis (2.5 μl) were then PCR amplified in TRAP buffer with all four dNTPs (50 μM each), 0.1 μg Cy5-labeled-TS primer, 0.1 μg ACX primer, TSNT primer mix, and 1 μl of Advantage cDNA Polymerase mixture (BD Biosciences Clontech, Mountain View, CA) in 50 μl of total reaction volume. TS primer labeled at the 5′ end with Cy5 was obtained from IDT (Coralville, IO). PCR amplification started with 94°C for 2 min followed with 36 cycles (30 sec at 94°C, 30 sec at 55°C, and 1 min at 72°C). All telomere repeat synthesis and PCR amplification reaction mixtures were supplemented with acetylated BSA to a final concentration 0.5 mg/ml (New England Biolabs, Ipswich, MA). The TRAP PCR products (1/3 of the total reaction per well) were separated on 7.5% acrylamide gels (ratio of acrylamide to bis-acrylamide 19:1). Gel images were captured using a Typhoon Trio imager in fluorescence mode (GE Healthcare, Waukesha, WI). Quantification was performed by Quantity One 1-D analysis software (Bio-Rad, Hercules, CA).
Terminal restriction fragment (TRF) length analysis
TRF analysis was performed as described previously . High-molecular-weight genomic DNA (3 μg) was digested with a cocktail of restriction enzymes (HinfI, AluI, HhaI, HaeIII, MspI, and RsaI) and separated in a 0.6% TBE agarose gel. Undigested and HindIII digested λ phage DNA was used as marker. DNA was Southern transferred to a Hybond-N+ membrane (GE Healthcare, Waukesha, WI) and hybridized at 42°C in Ultrahyb solution (Ambion, Austin, TX) to the telomeric probe (CCCTAA)6 end-labeled with [γ-32P]-ATP. Blots were washed under stringent conditions. Subsequently, the blots were rehybridized with a λ probe to visualize the position of the markers.
Fluorescence in situ hybridization of BAC clones
Standard fluorescence in situ hybridization (FISH) protocol on metaphase chromosomes derived from fibroblast cell lines was followed as described previously . Briefly, slides containing spread metaphase cells were treated with RNase (100 μg/ml/SSC, 30 min) and pepsin (10% pepsin in 0.01 M HCl, 10 min), and fixed in 1% formaldehyde/PBS/50 mM MgCl2. Slides where dehydrated in ethanol series followed by denaturation at 70°C with 70% (v/v) formamide/2 × SSC. Slides were dehydrated in an ethanol series and hybridized at 37°C overnight. The slides were washed in 50% formamide/2 × SSC (42°C), 2 × SSC (42°C) and 0. 1 × SSC (60°C) and stained with DAPI solution (10 μl DAPI (1 μg/ml) in 50 ml 2 × SSC) and mounted with vectashield solution (Vector Labs, Burlingame, CA). The platypus BAC clones CH236-606P3 (TERT) and OA_B_462c1 [GenBank:EU030443] (MHC) used for hybridization were obtained from CHORI (Oakland, CA). The clone OA_B_462c1 was previously mapped to X3Y3 . To investigate telomere repeats, a Cy3 primary conjugated telomere repeat (TTAGGG 42-mer) probe was used (Geneworks, SA, Australia).
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We thank M. L. Baker (University of New Mexico) for providing tissues of Monodelphis domestica, T. Daish and A. Casey (University of Adelaide) for help with the monotreme tissue collection, M. M. Lozano for providing BALB/c mice, and M. P. Domjan and N. Matthews for quail eggs (University of Texas, Austin). We thank W. Bargmann (University of Texas, Austin) for careful reading of the manuscript and T. Grant (University of New South Wales) and F. Carrick (University of Queensland) for helpful advice on platypus biology.
This study was supported by the Public Health Service grants from the National Cancer Institute (grant numbers CA33192, CA098151). F.G. is an Australian Research Council (ARC) Research Fellow.
The author(s) declare that they have no competing interests.
RH and JN carried out cloning, sequence characterization, RT-PCR, TRAP, TRF and synteny analysis, SLL prepared platypus RNA and tissue samples and performed FISH hybridization. FG and HRB supervised the work carried out in their laboratories and edited the manuscript. RH and JN conceived the study and wrote manuscript. All authors read and approved the final manuscript.
Electronic supplementary material
Additional file 1: PCR methods (PDF). (a) Primers used for identification of OanTERT and OanGAPDH expression. (b) PCR conditions. (c) Primers used for cloning of Oan. (PDF 30 KB)
Additional file 2: Platypus TERT cDNA clone (PDF). (a) Cloning strategy. (b) The sequence comparison of the genomic contig with the platypus TERT cDNA. (c) The assembled sequence of OanTERT. (d) The region of genomic sequence flanking at 3′ side the sequence matching OanTERT cDNA. (e) The region of genomic sequence flanking at 5′ side the sequence matching OanTERT cDNA. (PDF 6 MB)
Additional file 3: All these sequences, except Δ2p(136-end), can also be retrieved in text format from the Additional file 6. (a) Corrections of TERT protein sequence models. (b) De novo generated TERT protein sequence models. (c) Human AS TERT variant Δp2(136-end). (PDF 44 KB)
Additional file 4: Sequence alignment of platypus TERT with proteins of seven selected vertebrate species (PDF). The boundaries of the different domains and sequence motifs are indicated. This alignment serves as basis for the schematic representation of TERT proteins in Figure 1a. (PDF 25 KB)
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Hrdličková, R., Nehyba, J., Lim, S.L. et al. Insights into the evolution of mammalian telomerase: Platypus TERT shares similarities with genes of birds and other reptiles and localizes on sex chromosomes. BMC Genomics 13, 216 (2012). https://doi.org/10.1186/1471-2164-13-216
- Alternative splicing
- Sex chromosomes