Hrdličková R et al. (2012), Insights into the evolution of mammalian telome...

ECB-ART-50123

BMC Genomics 2012 Jun 01;13:216. doi: 10.1186/1471-2164-13-216.

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Insights into the evolution of mammalian telomerase: platypus TERT shares similarities with genes of birds and other reptiles and localizes on sex chromosomes.

Hrdličková R , Nehyba J , Lim SL , Grützner F , Bose HR .

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BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

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	Figure 1. Evolutionary relationship of platypus TERT. (a) Comparison of the protein domains and motifs in the platypus TERT protein (Oan) with seven TERT proteins of selected vertebrates: Hsa - human (Homo sapiens), Mmu - mouse (Mus musculus), Mdo - short-tailed opossum (Monodelphis domestica), Gga - domestic chicken (Gallus gallus), Aca - green anole (Anolis carolinensis), Xle - African clawed toad (Xenopus laevis), Tru - pufferfish (Takifugu rubripes). Different colors indicate the four different domains (TEN, TRBD, RT, CT), with the darker tone indicating conserved motifs within the domains. The names of the motifs are depicted under the platypus TERT schema. L1, L2, and L3 designate three linker regions of variable length and sequence. The two green filled circles show the boundaries of the regions encoded by the second TERT exon, including the putative second exon of platypus. (b) The percentage of similar amino acids at the same positions in the aligned sequences of TERT proteins of seven vertebrate species shown in panel (a) relative the platypus TERT protein. The percentages calculated relative to the length of platypus sequence are shown for separate conserved domains (TEN, TRBD, RT, and CT) and the full-length protein (TERT). TRBD domain sequence excludes sequences of linkers L2, L3. (c) Cumulative length of the three linker regions (L1, L2, L3) in the TERT proteins of different species is shown as percentage of the length of these regions in platypus TERT. The lengths were calculated based on the alignment of the TERT proteins of different species shown in Figure S3 and on the definition of the three linker regions shown in Figure 1a and Figure S2. The graph includes TERT proteins shown in panel (a) and following additional proteins: Ptr - chimp (Pan troglodytes), Mml - macaque rhesus (Macaca mulatta), Clu - dog (Canis lupus), Bta - cow (Bos taurus), Laf - African elephant (Loxodonta africana), Rno - rat (Rattus norvegicus), Mau - hamster (Mesocricetus auratus), Meu - wallaby (Macropus eugenii), Cja - Japanese quail (Coturnix japonica), Cmo - Muscovy duck (Cairina moschata), Tgu - zebra finch (Taeniopygia guttata), Cmi - elephant shark (Callorhinchus milii), Dre - zebrafish (Danio rerio), Nfu - killifish (Nothobranchius furzeri), Ola - medaka (Oryzias latipes), Ome - medaka (Oryzias melastigma), Eco - grouper (Epinephelus coioides), SpuL and SpuS - long and short form TERT of sea urchin (Strongylocentrotus purpuratus), Hro - leach (Helobdella robusta), Dpu - water flee (Daphnia pulex), and Hma - hydra (Hydra magnipapillata). Insect, nematode, and urochordate TERTs were not included because these proteins do not contain all four basal structural domains of the vertebrate TERT protein [38,57]. (d) Phylogenetic tree of vertebrate TERT proteins constructed by the Bayesian inference method. The construction is based on the alignment shown in the Additional file 5: Figure S3. For the abbreviation of protein species origin see the legend to panels (a) and (c). All these proteins were presumably full-length except the chimp (98% complete), opossum (99.5% complete), wallaby (97% complete), and elephant shark (approximately 80% complete). Sea urchin SpuL TERT was used to determine the root of the tree (indicated by the arrow, actual branch with SpuL not shown). Numbers shown at the corresponding branches are clade credibility values. Because most of the branches had clade credibility equal to 1.00, only those with the value <1.00 are shown. The long and medium linkers are indicated by squares by the abbreviated species names. The black squares indicate TERT proteins with long linkers (the L1, L2 and L3 linkers reaching 100%-160% of the length of platypus linkers). A TERT protein with the linkers of medium length (60%-99% of the platypus linkers) is marked by gray square.
	Figure 2. Platypus alternatively spliced TERT variants. (a) The comparison of platypus, chicken, and human AS variants. The figure show all identified platypus variants, and all chicken and human variants that involved the second exon. Additionally, the chicken and human variants most similar to platypus C variant are shown. The structure of wild-type (WT) platypus, chicken, and human TERT is shown at the top of each set of AS variants. The TERT ORFs are shaded. The positions of the exons and functional domains (TEN, TRBD, RT, CT) of TERT are indicated. The model of TERT transcripts with single alternative splice events are drawn to their relative sizes. Deletions (triangles) and insertion (gray rectangle) resulting from splicing events are indicated. The red triangles represent AS variants conserved between platypus and chicken. Blue triangle identifies AS TERT variant which contains a deletion of a similar RT region in platypus, chicken, and human TERT. The black triangles indicate AS variants which are not conserved among platypus, chicken, and human. Asterisks identify the location of the stop codons. The descriptive and trivial names of the TERT AS sequences are shown in the right margin. The numbers in the parentheses represent the position of the deletion in the exon which is indicated in front of these parentheses. The figure is based on data from the following GenBank entries of platypus isoforms: [GenBank:JF441069] (A), [GenBank:JF441066] (A3), [GenBank:JF441065] (A2), [GenBank:JF441068] (pA4), [GenBank:JF441070] (B), [GenBank:JF441067] (C), [GenBank:JF441072] (D); chicken isoforms: [GenBank:JF896279] (A2), [GenBank:DQ148473] (A), [GenBank:DQ681311] (A3), [GenBank:DQ256211] (V3), [GenBank:DQ148464] (G), and human isoforms: [GenBank:AB086379] (Δ7-8), and human isoforms [GenBank:JF896280] (Δ2), [GenBank:JF896283] (Δ2-8), [GenBank:JF896284] (Δ2-13). The available sequence of human Δ2p(136-end) variant is too short to be accepted by GenBank and is provided in the supplement (Additional file 3, p.5). (b) The ClustalX alignment of amino acid sequences encoded by the second exon of platypus (Oan), domestic chicken (Gga), Japanese quail (Cja), human (Hsa), and mouse (Mmu) TERT. The part of TEN domain present in the second exon is shown at the top. The novel SA and SD sites are indicated by small boxes. For mouse AS TERT variants see [GenBank:CF531121.1, GenBank:BC127068.1]. The prefixes to SD or SA represent species: Oan (platypus), ch (chicken), q (quail), h (human), and m (mouse). The name of the relevant platypus or chicken alternative splicing variant(s) is indicated after the SD and SA used to create this isoform(s). The residues shown by (*) are conserved in all species.
	Figure 3. Expression of TERT and its AS variants in various tissues. (a) The schematic representation of the PCR strategy for detection of OanTERT and its variants is shown. Arrows represent primers used for RT-PCR (Additional file 1: Table S1). The transcript spliced using 16 evolutionary conserved exons is designated as wild-type (WT) isoform. (b) The steady-state levels of TERT mRNA were determined by semiquantitative RT-PCR. The level of GAPDH served as a loading control. Aliquots from the PCR were taken every 5th cycle beginning with cycle 35 for the amplification of TERT and cycle 30 for GAPDH. The PCR products were resolved on agarose gels and visualized with ethidium bromide. Gray triangles indicate an increasing number of PCR cycles. (c) The expression of OanTERT AS variants was determined by RT-PCR (Additional file 1: Table S1). The position of PCR primers are shown in Figure 3a. The lower panel (7/8*) shows the PCR reaction with cDNAs obtained with synthesis using oligo-dT primer instead of random-hexamer priming. (d) The table summarizing the expression of OanTERT AS variants is shown. (e) Telomerase activity in extracts from different tissues of a male platypus (M1; lanes 1–9) and from brain, heart and skeletal muscle of three platypus females (F1, F2 and F3; lanes 11–19) was determined by the TRAP assay. All three animals evaluated were obtained from wild populations and were sexually mature adults, however, their precise age is not known. Telomerase activity in the same tissues was determined in three mouse females and one chicken female (lanes 20–31). Extraction buffer serves as the negative control (NC) (lanes 10 and 32). Molecular weights are indicated in the left margin and the positions of the amplified telomerase products (6 bp ladder starting with 50 bp band) and a 36 bp PCR internal control (IC) are shown in the right margin.
	Figure 4. Telomere length of the platypus. (a) Telomere length was determined by terminal restriction fragment (TRF) length analysis as described in Material and Methods section. This analysis included DNA isolated from quail, duck and chicken embryos, human (BD Biosciences Clontech, Cat. No. S0950), spleen from 2 month-old male mice (strain C57BL/6), lung from toad Xenopus tropicalis, opossum Monodelphis domestica, and platypus (M1, F1 and F2). The amount of DNA analyzed for F2 was higher (5 μg) than for all other samples (3 μg). The molecular weights (kb) are indicated on both sides of the picture. (b) Fluorescence in situ hybridization with Cy3 primary conjugated telomere repeat (TTAGGG 42 mer) probe (red signal), on female platypus metaphase spreads. Platypus chromosome 1 has large interstitial telomeric repeats. Scale bar = 10 μm.
	Figure 5. Chromosomal localization of the OanTERT locus. (a) Fluorescence in situ hybridization (FISH) of TERT-containing BAC clone on male platypus metaphase spread. Green fluorescence signals indicate the position of the TERT locus on a pair of metacentric chromosomes (green arrows). Scale bar = 10 μm. (b) FISH of TERT (green) and the major histocompatibility complex (MHC) (red) was performed as described in (a). TERT signals were identified on the pseudoautosomal regions of Y2qX3p. (c) Schematic representation of platypus chromosomes X2, Y2, X3, Y3. Approximate positions of TERT and MHC signals are indicated. Locations of gene contigs Ultra 474 (U474, blue), Ultra 43 and X3 (U43-X3, purple) are indicated as determined in this (c) and previous works [54,55]. (d) The analysis of shared synteny between TERT-containing chromosomal regions of platypus and human. The platypus genes located at platypus X2 and X3 chromosomes and their human orthologs (on human chromosomes) are indicated by distinct colored frames. The genes without the colored frame were not yet localized to platypus chromosomes. The numbers of the platypus gene contigs as present in current Ensembl database are indicated. “Ultra” in the designation of some contigs is shortened to “U”. The mutual orientation of platypus X3 and U43 contigs and their relative position on the X3 chromosome were not established so that the orientation shown in the panel C may not reflect their actual positioning and orientation. The genes located at different human chromosomes and their platypus orthologs (on platypus chromosomes) are indicated by distinct colored backgrounds. Light blue field identifies parts of human chromosomes that share synteny with chicken chromosome 2. Human genes located on 5p15 which have no platypus orthologs are indicated by gray lettering. The vertical arrows in the group of 6p24.3 genes and their platypus X2 orthologs represents a group of approximately 25 additional orthologous genes that extend shared synteny between 6p and X2.
	Figure 6. Telomerase activity in extracts from liver and heart of three platypus males and females. Telomerase activity was determined by the TRAP assay as described in Figure 3e. The column graphs under the lanes indicate the relative abundance of PCR-amplified telomerase reaction products determined by quantification of fluorescent signal.

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