Identification and characterization of bovine regulator of telomere length elongation helicase gene (RTEL): molecular cloning, expression distribution, splice variants and DNA methylation profile
- Zhuo Du†1,
- DingSheng Zhao†1, 2,
- YongHui Zhao1, 3,
- ShaoHua Wang1,
- Yu Gao1, 4 and
- Ning Li1, 4Email author
© Du et al; licensee BioMed Central Ltd. 2007
Received: 28 September 2006
Accepted: 06 March 2007
Published: 06 March 2007
The genetic basis of telomere length heterogeneity among mammalian species is still not well understood. Recently, a gene named regulator of telomere length elongation helicase (RTEL) was identified and predicted to be an essential participant in species-specific telomere length regulation in two murine species. To obtain broader insights into its structure and biological functions and to ascertain whether RTEL is also a candidate gene in the regulation of telomere length diversity in other mammalian species, data from other mammals may be helpful.
Here we report the cDNA cloning, genomic structure, chromosomal location, alternative splicing pattern, expression distribution and DNA methylation profile of the bovine homolog of RTEL. The longest transcript of bovine RTEL is 4440 nt, encompassing 24.8 kb of genomic sequence that was mapped to chromosome 13q2.2. It encodes a conserved helicase-like protein containing seven characterized helicase motifs in the first 750 aa and a PIP box in the C-terminus. Four splice variants were identified within the transcripts in both the coding and 5'-untranslated regions; Western blot revealed that the most abundant splice variant SV-1 was translated to a truncated isoform of RTEL. The different 5'UTRs imply alternative transcription start sites in the promoter; Bovine RTEL was transcribed at the blastocyst stage, and expression levels were highest in adult testis, liver and ovary. DNA methylation analysis of tissues that differed significantly in expression level indicated that relatively low DNA methylation is associated with higher expression.
In this study, we have identified and characterized a bovine RTEL homolog and obtained basic information about it, including gene structure, expression distribution, splice variants and profile of DNA methylation around two putative transcription start sites. These data may be helpful for further comparative and functional analysis of RTEL in mammals.
Telomeres are specialized nucleoprotein complexes that form the natural ends of eukaryotic chromosomes and have important structural and protective roles. Cellular telomere length, which undergoes dynamic changes and is stringently regulated by a series of telomere-associated proteins, is fundamental to the understanding of cell growth and survival, replicative life-span and carcinogenesis [1–3]. In recent years, several factors other than the well-known telomerase regulation pathway have been shown to regulate telomere length considerably : oxidative damage [5, 6] or mutation of the telomeric DNA , high-order alternative structures, especially the G-quadruplex organized from guanine-rich regions [8, 9], the T-loop structure of the telomere , illegitimate DNA recombinations and inappropriate repair of telomeric DNA all influence telomere length . Recently, a novel gene named regulator of telomere length elongation helicase (RTEL), which encodes a helicase-like protein with several functional helicase domains/motifs, was shown to participate in telomere length regulation in two murine species [9, 32]. By incorporating phenotypic data from RTEL knock-out ES cells and comparing the function of a nematode homolog gene named dog-1 (deletion of G tracts) , RTEL is predicted to unwind "harmful" structures that form in the G-rich region of genome, especially the telomeric DNA, in order to protect the telomere and influence the length balance . As a potential G-quadruplex resolvase, RTEL plays an important role in telomere maintenance, embryonic development and survival in mice. At the same time, as a safeguard for the genome, it contributes to the maintenance of genetic stability by minimizing unexpected recombinations induced by G-quadruplex.
The genetic basis of telomere length heterogeneity among mammalian species is still largely unknown. RTEL was identified as an essential regulator of telomere length in mice, in order to obtain broader insights into its biological functions, information from other mammalian species may be helpful. It may be essential for regulating telomere length diversity in mammals in general, but little information has become available to date. Here we describe some basic information about a bovine RTEL homolog, including the genomic organization, cDNA sequence, expression distribution, forms of splice variant and status of DNA methylation, which may provide useful data for further comparative analysis. In addition, since RTEL could protect the telomere as well as prevent genome instability, both of which are critical for cell survival and longevity, RTEL may be a candidate for extending the replicative potential and life-span of cultured cells. Replicative senescence and the proliferative limit of in vitro cells is a major obstacle to the genetic manipulation of farm animals.
Identification and characterization of bovine RTEL
Using bioinformatics tools, 11 overlapping EST sequences [GenBank: BE753036, DV890221, DV869581, DV923036, DV875515, BM031944, DV819111, DN514403, BI539060, CK947294, BE663182], which share significant homology to human and mouse RTEL, were identified and assembled. Together with genomic sequence information partly released by the Bovine Genome Project, this enabled us to obtain a 4.3 kb sequence encoding a protein with high similarity to the human and mouse homologs. Using two sets of primers, the coding region of bovine RTEL was amplified in two overlapping fragments (1.9 kb and 2.1 kb respectively) by RT-PCR. After sequencing and comparison with mouse and human RTEL, the coding region of bovine RTEL was identified. To determine the transcription start site and polyadenylation site, 5' and 3' RACE were performed: a 280 bp 3'RACE amplicon and two forms of 5'RACE amplicons (approximate 400 bp and 250 bp respectively) identical to the coding sequence were obtained by nested PCR. The full-length cDNA was then obtained by incorporating the coding and 5'/3'-untranslated regions. The longest transcript of bovine RTEL [GenBank: DQ323153] is 4440 nt, containing a 3801 nt major open reading frame flanked by a 257 nt (or alternatively 137 nt) 5'UTR and a 383 nt 3'UTR; the polyadenylation signal AAUAAA was identified 17 nt upstream of the poly(A) homopolymer. The nucleotide sequence of the coding region shows high identity with the human and mouse homologs: 70.9% with mouse RTEL [GenBank: NM_001001882], 76.6% with human NHL  (human homolog of RTEL [GenBank: NM_016434]; mouse and human show 77.1% identity with each other.
Genomic organization of bovine RTEL
Exon size (bp)
Intron size (bp)
In summary, these comparisons of the coding and deduced protein sequence, genomic organization and domain/motif structure indicate that we have identified the bovine homologue of RTEL.
Chromosomal location of bovine RTEL
Alternative splicing of bovine RTEL
The full-length RTEL cDNA and splice variant isoforms 1 and 2 are detected in high abundance in all the tissues examined; SV-3 is relative rare (2 out of 31) and is tissue-specific (detected only in testis). Since all three alternative splice variants are located in the coding region, there are considerable changes in the deduced protein sequences. The differences between the splice variants and wild type (the protein sequence deduced from the longest transcript) at the protein level are shown in figure 3D. For SV-1, the exon 31 truncation leads to a frame shift at codon 1063 and an early translation stop at codon 1084, generating a shorter peptide of 1083 aa lacking the PIP box. For SV-2, because the nucleotide lost is 3N, only 29 aa are lost followed by a Glu to Thr conversion without frame shift. Like SV-2, SV-3 lacks 50 aa and causes an Asp to Arg conversion.
To determine whether the first 21 exons also undergo alternative splicing, RT-PCRs were performed with primers covering exons 2 to 25 using pooled cDNA prepared from testis, ovary, liver and spleen. After 30 cDNA clones were sequenced, no alternatively spliced isoform was identified in this region. Therefore, like in mouse and human, alternative splicing of bovine RTEL is only detected in the last 5 exons.
Apart from the coding region variations, we also identified two forms of 5'UTR, named 5'UTR-1 and 5'UTR-2 [GenBank: DQ420364], by 5'RACE; 5'UTR-1 is the more commonly used form (10 of 16 clones). RT-PCRs were carried out using primers based on the differential region to verify that both forms of 5'UTR existed in the transcripts. The two forms were indeed confirmed by sequencing, at least in testis. Further analysis of the genomic sequence identical to the 5'UTR indicates that each form of 5'UTR is generated by selectively utilizing two of the three putative exonic regions, which is consistent with GT-AG rule: 5'UTR-1 selects the second and third exon-like regions, whereas 5'UTR-2 links the first and parts of the third exon-like regions by skipping the second one (figure 3B). Consequently, the 5'UTR sequences differ significantly, sharing only 55 nt in the region flanking the ATG site. Two mechanisms may be responsible for producing the different 5'UTRs: alternative splicing can explain the skipping of part of the third exonic region in 5'UTR-2 because an alternative AG acceptor site is observed in this region; whereas the selective utilization of the second exonic region in 5'UTR-1 may involve an alternative promoter or alternative transcription start site (TSS). Genes containing splice variants of this kind (figure 3B) usually have alternative promoters or, more exactly, alternative TSS. So on the basis of the different 5'UTRs, we presume that there are two TSSs for bovine RTEL transcription.
Overall, four splice variants were identified and characterized in bovine RTEL transcripts, three in the coding region and one in the 5'UTR. Among the coding region splice variants, SV-1, the most abundant isoform, encodes a prematurely truncated protein lacking the PIP box.
Expression analysis of bovine RTEL
Since telomeres progressively shorten during cell division and senescence, we further compared the expression level of bovine RTEL between normal and senescent cultured cells. The expression data suggest that in cultured cells, the expression level of RTEL is very slight; and no significant difference was observed between normal and senescent cells. (figure 4C). The senescent cell phenotype was confirmed by a β-galactosidase activity assay (data not shown).
DNA methylation analysis
In this study, we have identified the bovine homolog of RTEL and provided some basic information including sequence, genomic organization, chromosomal location, expression pattern, splice variants and DNA methylation profile. From the mRNA and protein sequences, RTEL is to some extent conserved between bovine and mouse. Four splice variants were characterized in both the 5'UTR and the coding region, resulting in extensive changes in the protein sequence including amino acid substitutions and deletions, frame shifts and translation stops in the carboxy-terminus (figure 3). The high abundance of splice variants of RTEL in mouse, bovine and human indicates its potential functional importance. In mouse, splice variants may be an important clue to explain the telomere length difference between Mus musculus and Mus spretus . As for the relative abundances of the splice variants, SV-1 and SV-2 are detected in the mRNA from all the tissues analyzed in relatively high abundance and show no tissue specificity, whereas SV-3 has low abundance and is only detected in testis. Its low abundance may result from tissue-specific alternative splicing. It is well known that many low-abundance splice variants may also contribute greatly to gene function because of the temporal specificity of splicing  or other unknown amplifying mechanism. At the protein level, all these splice variants produce novel C-terminus with low or no homology to the wild-type bovine RTEL, increasing the protein diversity. In addition, as alternative splicing occurs mainly in the last 5 exons, resulting in considerable amino acid changes in the region where few functional motifs/domains have been identified (750 aa-1266 aa), it also provides a good way to investigate the functions of this region. SV-1, where the PIP box is absent from the protein sequence, would be helpful for investigating the function of PIP and the functional relationship of RTEL and PCNA, and we have demonstrated that SV-1 indeed translated to a truncated protein by Western blot.
In mouse, five coding region splice variants were detected and identified in the last four exons (29 to 34) . Some alternative splicing patterns are conserved between mouse and bovine: splice variant 4 in mouse resembles SV-1 and SV-2 in bovine. But our data show many differences from mouse: in the protein sequence, frame shift and early translation stop variants were observed; and one tissue-specific splice variant (SV-1) was identified (only detected in testis). Besides the coding region splice variants, we also identified two significantly different forms of 5'UTR (5'UTR-1 and 5'UTR-2). The data suggest that there are alternative transcription start sites in the promoter region. The organization and transcriptional regulation of RTEL is more complex than expected. Thisstudyprovides only a preliminary analysis of alternative splicing, It is still unclear how many of the splice variants we identified are functional and how many are aberrant ('noise'). Some splice variants may be by-products of false pre-mRNA splicing, so further analysis at the protein level and the biochemical properties of each variant is necessary. The functional significance of the different 5'UTR isoforms is also an interesting question for further investigation.
Heterogeneity of telomere length is an important branch of the exciting field of telomere biology, many useful data have been provided by previous studies, paving the way for further revelations about the underlying mechanism. In the present study, we have identified and characterized the bovine homolog of RTEL, obtaining some basic information including expression pattern, alternative splicing and DNA methylation profile of the putative promoter region. All these data may be useful for further functional analysis and comparison of RTEL between bovine and other mammalian species. This would contribute to further understanding of the role of RTEL in regulating species-specific telomere length diversity. At the same time, in view of the capacity of RTEL to protect telomeres and maintain chromosome stability, this gene is also a candidate for extending the proliferative life span of cultured cells and preventing genomic instability, which currently vitiates the precise genetic manipulation of farm animals.
In a previous study, RTEL was identified and functionally validated as an essential gene in the regulation of species-specific murine telomere length. However, little information is available about other mammalian species. In this article, we describe some basic information about bovine RTEL, with a view to providing useful data for further comparative and functional analysis. We have identified the bovine homolog and provided some comparative structural and functional information. Comparisons of the coding and deduced protein sequence, genomic organization and domain/motif structure indicate that RTEL is conserved between bovine and mouse. The expression patterns and modes of alternative splicing are also similar. The deduced promoter region of bovine RTEL lacks the typical TATA box and CAAT box, but presents a relatively high density of GC box and a high GC content, which is also seen in mouse. DNA methylation analysis indicates that in the tissues examined, ignoring differences in expression, the overall DNA methylation level is lower in the deduced promoter region; however, relatively low methylation was observed in tissues with high expression levels. To obtain broader insights into the biological functions of RTEL in mammals, our work may facilitate further understanding of the function of RTEL in bovines and elucidate whether RTEL is a candidate for regulating telomere length diversity in all mammalian species.
RNA preparing and gene cloning
Tissues (heart, liver, spleen, lung, kidney, cardia, muscle, ovary, and testis) were collected from adult cattle and were immediately stored in liquid nitrogen. All the experimental research on animals was follow internationally recognized guidelines. Total RNA was extracted from the above mentioned tissues using Trizol reagent (Invitrogen) according to recommendations of the manufacturer. Approximate 1 μg of total RNA was used for the first-strand cDNA synthesis (M-MLV reverse transcriptase, Promega) with d(T)18 primer. Two sets of primers (F1-5'CCC AAG AGT AGA CCA GTA TGC C, R1-5'ACA CGG ACA CTG AGA CAA TGC; and F2-5'CGG AGG TGA TGG AGG CTT TC, R2-5'AGT TCT GGG CAA GCA CAT TCC) were used to amplify bovine RTEL in two overlapping fragments. All the PCRs were performed in 25 μL containing 2 μL of cDNA, 2.5 μL of PCR buffer (Mg2+ plus), 200 μM each dNTP mixture, 10 pM of each primer and 1 U Taq DNA polymerase. PCRs were run for 35 cycles of 94°C for 30s, 60°C for 30s and 72°C for 2.5 min, followed by incubation at 72°C for 10 min. The resulting PCR products were gel-purified and subsequently ligated into pMD18-T vector (TaKaRa) and sequenced. For each amplicon, three individual clones were sequenced on both strands. The primers used for cDNA cloning were also used for the detection of splice variants.
Rapid amplification of cDNA 3' and 5' end
Total RNA from testis was used for 3' and 5'RACE using the 3'/5'-RACE kit (Invitrogen). For 3'RACE, first-strand cDNA synthesis was performed using the adapter primer (AP), then two rounds of 3'RACE-PCRs were performed using the abridged universal amplification primer (AUAP), and gene specific primer 1(GSP1) for first round; AUAP and GSP2 were used for the nested PCR. (3GSP1-5'GTG CCC TAC CTT GCC GAT GTC CG, 3GSP2-5'CCC TGA GAT GGC AGT GGG A). The PCRs was performed using the following conditions: 35 cycles of (94°C for 30s, 50°C for 30s and 72°C for 1 min) for first PCR; and 35 cycles of (94°C for 30s, 58°C for 30s and 72°C for 1 min) for nested PCR.
For 5'RACE, three nested GSP primers (5GSP1-5'CAC TGT TGT CTC CAG GCA GCT C, 5GSP2-5'GTA GCA GGC TGG GAC ATC TC, 5GSP3-5' CCT GCA GAC ACT CCA AAA CC) were designed on the basis of coding sequence. Primer 5GSP1 was used for first-strand cDNA synthesis at 42°C for 60 min; then cDNA was purified and added a homopolymere dC tail following the instructions of invitrogen 5' RACE kit. The following conditions were used for 5'RACE-PCRs: Abridged anchor primer (AAP) and 5GSP2 were used for first PCR in 35 cycles of (94°C for 30s, 50°C for 30s and 72°C for 1 min); AUAP and 5GSP3 were used for nested PCR in 30 cycles of (94°C for 30s, 58°C for 30s and 72°C for 45s). The PCR products were sequenced after gel purification and cloning.
Amplification and sequencing of genomic region
Genomic DNA was extracted from blood using standard phenol protocol. To amplify RTEL, long-range PCRs (TaKaRa LA Taq™) were performed using five sets of primers complement to the exonic regions. All PCRs were performed by 35 cycles of 98°C for 10s and 68°C for 5 min, the resulting products were gel-purified and ligated into pMD18-T vector (TaKaRa), and sequenced by primer walking. The primer sets for long-range PCRs were as follow: (F1-5'ACA GGG AAG ACA CTG TGC CTT CTC, R1-5'CTG CTA CTT CAG GTT CCG AGA CAG; F2-5'TAC TAC CTG TCT CGG AAC CTG AAG, R2-5'GCT TGG TGA CAC CAC TGT TGT CTC; F3-5'GAG ACA ACA GTG GTG TCA CCA AGC, R3-5'TGT TCC AGA CAT CTG ATC GCT GAG; F4-5'AGG ACA GCT CAG CGA TCA GAT GTC, R4-5'CAA GGT CAT CGG AGC TCT TGT AGT; F5-5'GTC CCT CCC CAC CTA ACC TAA C R5-5'AGA GTC GAG TGG TCC CAG TTC G).
Chromosomal location of bovine RTEL
Bovine 5 k (90 hybrids) radiation hybrid panel provided by Texas A&M University was used to determine the chromosomal location of RTEL. PCR screening was performed using the primer F-5'AAG TGA ACG GCA TTC TGG AG and R-5'CTG GGA CAT CTC CTT CCG GG. The results were submitted to BOVINE RADIATION HYBRID MAPPING and analyzed by RHMAPPER-1.22, The method of fluorescence in situ hybridization (FISH) was modified from Coppieters W et al. Briefly , Probe was labelled with biotin-14-dATP (Invitrogen) following the standard protocol, and coprecipitated with 50-fold excess of salmon sperm DNA. Hybridization was performed for 48 hours at 37°C in a humid chamber. Chromosomes were counterstained with 0.5 μg/mL propidium iodide. Images were taken with epifluorescence microscope equipped with a DP70 CCD camera (Olympus, Japan), and fluorescence signals were enhanced by software Video TesT-FISH (Video TesT Ltd, Russia, 2003). The chromosome slides were treated in 0.01% trypsin (Gibco) at 37°C for 50 seconds, and then stained with 0.04% Giemsa solution for G-binding.
Embryo lysis and cDNA amplification
Four cloned blastocysts were used for preparing the template for expression analysis by protocol modified from those previously used . PCRs were performed in standard condition: 35 cycles of 94°C for 30s, 58°C for 30s and 72°C1 min using primers F-5'CCA CAA GCA GCA GTT TGA GG and R-5'CAT GTT CAC TGG GGC TCT GG, the same primers were used for the second-round PCR under the same condition.
Approximately 20 μg of total RNA extracted from adult tissues and fibroblast cells were size-fractioned in 1% formaldehyde-denatured agarose gel. Northern blot was performed following standard protocol, and α32-P labeled 2 kb amplicon of bovine RTEL cDNA and 600 bp region of housekeeping gene GAPDH were used as probes.
Antiserum preparation and Western blot
cDNA region of wild-type RTEL, RTEL splice variant 1 and RTEL767–1060 were cloned into prokaryotic expression vector PET-30a (Novagen), all the inserts were verified by sequencing and transferred into BL21-Rosetta (DE3). The expressions of his-tagged recombinant proteins were induced by 0.1 M IPTG. The RTEL767–1060 peptide was purified by Ni-NTA resin (QIAGEN) and used to prepare rabbit antiserum. The homogeneous of purified RTEL767–1060 was >90% according to the result of SDS-PAGE. To prepare anti-RTEL767–1060 antiserum, two NZW Specific Pathogen-free (SPF) rabbits were immunized with the recombinant protein purified following standard procedure. Western blot was performed using anti-RTEL polyclonal antibody and anti-beta-actin antibody following the standard protocol.
Genomic DNA were modified by bisulfite based on previously described protocol [30, 31]. -Twentieth of the treated template was used for PCRs in standard condition. To amplify each CpG region, we used 35 cycles of 94°C for 30s, 50°C for 30s and 72°C 30s for the first PCR; and 30 cycles of 94°C for 30s, 57°C for 30s and 72°C 30s for the hemi-nested PCR. Primers used for amplifications were listed as follows: For CpG region1: region1 F1 (5'AGT TGT TTG AGA GGG ATT TGG) and region1 R (5'TCT TCC CTA AAC TCT CTA CAC T) for first PCR, region1 F2 (5'GGG ATG ATT TTG AGG AGG GAG T) and region1R for hemi-nested PCR; the primers for CpG island 2 were: region2 F (5'GGA TGG AGA TGG TTT AGA GAA T) and region2 R1 (5'CAA ACC ACT TCA TCA ATT CCC) for first PCR, region2 F and region2 R2 (5'CCT ATA ACA TCC CAT CAC AA) for hemi-nested PCR; as for amplify CpG region control, primers regionC F1 (5'GGA GAGTT TTA TAG GTA TAG GGA AG) and regionC R1 (5'CCC ACC TCC CTA CCA AAA GTA AAA C) were used for first PCR and regionC F2 (5'AGG TAT AGG GAA GAT ATT GTG TTT T) and regionC R2 (5'AAA ACA TCA CCT AAA ACA TCT CCT T) for nested PCR. The resulting PCR products of each CpG islands was gel-purification and ligated into pMD18-T Vector (TaKaRa), and 10 random individual positive clones were sequenced.
List of abbreviations
Regulator of Telomere Length elongation helicase
Proliferating Cell Nuclear Antigen
- RH mapping:
Radiation Hybrid mapping
Fluorescence In Situ Hybridization
Transcription Start Site
This work was supported by the National Natural Science Foundation of China and National Major Basic Research Program of China. We are grateful to Dr. James Womack (College of Veterinary Medicine, Texas A&M University) for providing the bovine 5 k (90 hybrids) radiation hybrid panels and to Li Lin for sharing the experience of DNA methylation analysis. BAC clones were provided by Institut national de la recherche agronomique (INRA) resource center, 78352 Jouy-en-Josas Cedex, France. We thank 'Manuscript Presentation Service' for English improvement of the manuscript.
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