Medaka tert produces multiple variants with differential expression during differentiation in vitro and in vivo

Abstract

Embryonic stem (ES) cells have immortality for self-renewal and pluripotency. Differentiated human cells undergo replicative senescence. In human, the telomerase reverse transcriptase (Tert), namely the catalytic subunit of telomerase, exhibits differential expression to regulate telomerase activity governing cellular immortality or senescence, and telomerase activity or tert expression is a routine marker of pluripotent ES cells. Here we have identified the medaka tert gene and determined its expression and telomerase activity in vivo and in vitro. We found that the medaka tert locus produces five variants called terta to terte encoding isoforms TertA to TertE. The longest TertA consists of 1090 amino acid residues and displays a maximum of 34% identity to the human TERT and all the signature motifs of the Tert family. TertB to TertE are novel isoforms and have considerable truncation due to alternative splicing. The terta RNA is ubiquitous in embryos, adult tissues and cell lines, and accompanies ubiquitous telomerase activity in vivo and in vitro as revealed by TRAP assays. The tertb RNA was restricted to the testis, absent in embryos before gastrulation and barely detectable in various cell lines The tertc transcript was absent in undifferentiated ES cells but became evident upon ES cell differentiation, in vivo it was barely detectable in early embryos and became evident when embryogenesis proceeds. Therefore, ubiquitous terta expression correlates with ubiquitous telomerase activity in medaka, and expression of other tert variants appears to delineate cell differentiation in vitro and in vivo.

Keywords: TRAP; medaka; pluripotency; senescence; telomerase; tert.

Figure 1

Medaka tert sequence. Top, Nucleotide sequence of medaka terta. Bottom, Deduced amino acid sequence of medaka TertA. Sequences of different exons are shown alternatively in black and blue color. ATG, TGA and a putative polyadenylation signal are shown in bold. Primer sequences used for RT-PCR analyses are underlined. Sequences absent in tertb and TertB are highlighted in grey.

Figure 2

Tert sequence alignment. Conserved motifs are boxed. Ol, Oryzias latipes (medaka); Fr, Takifugu rubripes (pufferfish); Xl, Xenopus laevis (clawed frog); Mm, Mus musculus (mouse); Hs, Homo sapiens (human). For accession numbers see Figure 3.

Figure 3

Tert protein, gene structure and chromosome context. (A) Phylogenetic tree. The bootstrap value is 100 at all branches. Sequence accession numbers follow organisms. (B) Domain structure of Tert proteins and cloning steps. The N-terminal, central and C-terminal regions are defined. Four conserved vertebrate motifs (v1-v4) and the telomerase-specific motif (T) in the N-terminal region, seven reverse transcriptase motifs (1, 2, A, B, C, D and E) in the central region, and three conserved motifs (v5-v7) in the C-terminal region are boxed. Sizes of cDNAs and proteins, predicted molecular weights and isoelectric point (pI) are shown in boxes. The difference in protein size is due mainly to the variable N-terminal region, in particular the linker between motifs v1 and v2. Note the medaka protein is the smallest among vertebrate proteins so far reported. Cloning steps are illustrated above the medaka Tert protein. Positions of primers used for cloning are indicated, their sequences are listed in Table 1. (C) Comparison of tert gene structure between medaka and human. The primary tert transcript is 4 times larger in human than in medaka. The human introns are not proportional. (D) Chromosome synteny between medaka and human. Chromosomal locations are shown in parentheses.

Figure 4

RNA variants and protein isoforms. (A) Genomic organization. Filled box, translated exon; open box, untranslated exon; thin line between boxes, intron. Exons are numbered within boxes, and sizes of introns are given between boxes. Asterisks denote stop codons. Arrows depict PCR primers. (B) Medaka isoforms TertA to TertE. Different domains are highlighted in different colors as for their encoding exons shown in (A). Sequences have been deposited in the Genbank under accession numbers DQ248968 (TertA) and JF326825-JF326828 (TertB-E).

Figure 5

RNA expression and telomerase activity. (A-C) tert RNA expression in adult tissues (A), developing embryos (B) and cell cultures (C). Numbers of PCR cycles are indicated. MES1, medaka ES cells; SG3, adult medaka spermatogonial stem cells; Or1 and Sok, uncharacterized cell cultures from the adult medaka testis; neg, negative control by using total RNA instead of cDNA as a template. An equal amount of cDNA reaction was used for each sample. β-actin was used as a loading control. (D-F) Telomerase activity in adult tissues (D), developing embryos (E) and cell cultures (F). Positive (pos; +) and negative controls (neg; -) were TRAP reactions by using the commercially supplied extract without and with heat inactivation. S125 and i1, uncharacterized cell cultures from medaka embryos of strain SOK and adult medaka testis of strain i¹. (G-J) Phenotypes of representative cell lines. Scale bars, 50 μm.

Figure 6

RNA expression of tert variants during differentiation in vitro and in vivo. (A) RNA expression during ES cell differentiation. Notably, expression of variant tertc increases upon ES cell differentiation. (B) RNA expression in representative stages of embryos and adult tissues. Elevated expression of variant tertc and tertd is seen in embryos at 12 h and 24 h post fertilization when overt cell commitment and differentiation take place. nanog and ntl (no tail) were used as markers of pluripoteny and differentiation, respectively. β-actin was used as a loading control. Arrows define PCR primers as illustrated in Figure 4A.