Abstract
Tardigrades, also known as water bears, are small aquatic animals that inhabit marine, fresh water or limno-terrestrial environments. While all tardigrades require surrounding water to grow and reproduce, species living in limno-terrestrial environments (e.g. Ramazzottius varieornatus) are able to undergo almost complete dehydration by entering an arrested state known as anhydrobiosis, which allows them to tolerate ionic radiation, extreme temperatures and intense pressure. Previous studies based on comparison of the genomes of R. varieornatus and Hypsibius dujardini - a less tolerant tardigrade - have pointed to potential mechanisms that may partially contribute to their remarkable ability to resist extreme physical conditions. In this work, we have further annotated the genomes of both tardigrades using a guided approach in search for novel mechanisms underlying the extremotolerance of R. varieornatus. We have found specific amplifications of several genes, including MRE11 and XPC, and numerous missense variants exclusive of R. varieornatus in CHEK1, POLK, UNG and TERT, all of them involved in important pathways for DNA repair and telomere maintenance. Taken collectively, these results point to genomic features that may contribute to the enhanced ability to resist extreme environmental conditions shown by R. varieornatus.
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Introduction
Tardigrades are small animals classically included in the clade Panarthropoda, together with Arthropoda and Onychophora. More than 1,200 species of tardigrades have been reported to inhabit all kinds of water environments. Even though they require surrounding water to grow and reproduce, limno-terrestrial tardigrades are well known for their remarkable capacity to endure extreme circumstances (such as dehydration, radiation, high and low temperature, high pressure, heavy metals and even outer-space conditions) when entering the anhydrobiotic state1,2,3,4,5,6. Nevertheless, some marine tardigrade species, such as Echiniscoides sigismundi, also present the ability to resist extreme dessication and intense gamma radiation7,8. Studies focused on survival and reproduction indicate that R. varieornatus presents a longer lifespan than H. dujardini5.
The study of the genomic sequence of one of the most stress-tolerant limno-terrestrial tardigrade species, R. varieornatus, has reported genomic alterations such as the expansion of several stress-related genes and the selective loss of peroxisomal oxidative and autophagy-related pathways, which can contribute to their tolerance to extreme environmental conditions9. Parallel studies have addressed the genome characterization of freshwater tardigrades, such as H. dujardini, which are among the least desiccation-resistant members of the phylum Tardigrada10, since they require previous conditioning to desiccation before entering anhydrobiosis. Such studies have also revealed various modifications in genes involved in macromolecule protection and stress signaling pathways that could contribute to the biological features exhibited by this tardigrade species, which lacks the extreme tolerance of R. varieornatus11. Other genomic comparative analyses have previously contributed to elucidate the mechanisms underlying aspects such as cancer resistance or longevity in different species11,12,13,14,15,16.
These genomic data have also revealed in R. varieornatus the presence of a novel tardigrade-unique protein called Dsup (damage suppressor) that suppresses X-ray-induced DNA damage and improves radiotolerance9. Nonetheless, recent studies found a Dsup homologue in H. dujardini that, despite its weak similarity with R. varieornatus Dsup, also presents nuclear localization and similar profiles in hydrophobicity and charge distribution along the protein17. This finding suggests that additional factors are involved in R. varieornatus extraordinary resistance to extreme conditions in comparison to H. dujardini, therefore encouraging the search for new hypotheses that explain the extremotolerance differences shown by these tardigrade species.
In this work, we have further explored the molecular mechanisms conferring extreme tolerance to limno-terrestrial tardigrades by comparing the genomes of R. varieornatus and H. dujardini, as well as that of a distant arthropod (Drosophila melanogaster). To this purpose, we have performed exhaustive manual annotation in these genomes of more than 250 genes involved in different DNA repair mechanisms. This comparative genomic analysis, together with the experimental validation of the identified alterations, has allowed us to detect specific gene amplifications and residue alterations in proteins involved in DNA repair pathways that may contribute to the enhanced tolerance to extreme environments exhibited by R. varieornatus.
Methods
Gene selection
Prior to genome annotation, we curated a list of more than 250 genes involved in oxygen homeostasis, stress response, telomere maintenance and DNA repair. Each gene was selected based on the experience of our laboratory in these fields18,19,20,21,22,23, and following a detailed revision of the available publications on each subject.
Genomic analysis
We performed manual annotation of genomes H. dujardini (assembly 3.1, GCA_002082055.1) and R. varieornatus (assembly 4.0, GCA_001949185.1) using the BATI algorithm (Blast, Annotate, Tune, Iterate)24, that allows researchers to annotate the coordinates and intron/exon boundaries of genes in novel genomes from Tblastn results. This procedure also enables the user to identify novel homologues. In addition to each genome, the algorithm was fed reference sequences from D. melanogaster and automatically-annotated H. dujardini (obtained from Ensembl and NCBI databases). This supporting information contributes to generate homology-based alignments that are later interpreted and revised manually, thus allowing the researcher to apply the experience in defying genes and obtaining better and more precise genomic structures (especially in the case of the aforementioned exon/intron boundaries). Once the selected genes were properly annotated, we compared the resulting sequences of R. varieornatus and H. dujardini to those of human, chimpanzee (Pan troglodytes), mouse (Mus musculus), naked mole rat (Heterocephalus glaber), dog (Canis lupus familiaris), chicken (Gallus gallus), zebrafish (Danio rerio), Japanese rice fish (Oryzias latipes), coelacanth (Latimeria chalumnae), fruit fly (D. melanogaster) and roundworm (Caenorhabditis elegans) when available. This allowed the identification of gene expansions and losses, as well as residue changes specific of R. varieornatus and H. dujardini. In the alignment of TERT, we also included the HIV-1 RT sequence. In the alignment of POLK, we also included the prokaryotic species Bdellovibrio bacteriovorus, Clostridium tetani, Escherichia coli, Mesorhizobium japonicum and Mycobacterium tuberculosis. We evaluated the putative effects of these residue changes using data from NCBI Conserved Domains, UniProt and ClinVar databases.
PCR analysis
To validate copy-number variations of genes of interest that we obtained through manual annotation, we performed PCR reactions with primer pairs that amplified a target region of the genomes of R. varieornatus and H. dujardini with different nucleotide sequences in each copy (Supplementary Table 1), and then examined the resulting electropherogram for evidence of both copies. R. varieornatus tardigrades were kindly provided by Dr. Takekazu Kunieda, University of Tokyo, Japan, while H. dujardini tardigrades were obtained from Sciento. Samples consisted of 50 tardigrades per species, which were snap frozen with liquid nitrogen. DNA was extracted using the QIAamp DNA Micro Kit (Qiagen). We tested the success of the PCR reactions by electrophoresis of the resulting products in a 1.5% agarose gel. Finally, the products were sequenced using the Sanger method and an ABI PRISM 3130xl Genetic Analyzer (Thermofisher). The results of the manual annotation and PCR analysis were also confirmed through RNA-Seq data from H. dujardini and R. varieornatus present into the NCBI Sequence Read Archive (SRA).
Homology models
Homology models of selected proteins were performed with SWISS-MODEL25 and used to evaluate the potential function of the residues analysed in this manuscript. The sequences of CHK1 and POLK from R. varieornatus were modelled using structure 1jx4 and 3jvr as a template, respectively. Similarly, the sequence of UNG from R. varieornatus was modelled using structure 1q3f as a template. The resulting structure was aligned to structure 1ssp to study its putative mode of interaction with a DNA substrate. The results were inspected and rendered with DeepView v4.1.0. Electric potentials were calculated with DeepView using the Poisson-Boltzmann computation method. Figures were generated with PovRay (http://povray.org) and UCSF Chimera26.
Results and Discussion
Manual annotation of genes involved in DNA repair, stress response, telomere maintenance and oxygen homeostasis in tardigrades
To study the molecular mechanisms linked to the increased resistance to extreme environmental conditions shown by the tardigrade species R. varieornatus in comparison to H. dujardini, we analyzed a set of more than 250 genes involved in stress response, oxygen homeostasis, telomere maintenance and DNA repair (Table 1). Manual annotation of this gene set allowed us to find copy-number variations in genes related to DNA repair pathways, as well as to verify the previously described variations for both species of tardigrades. Interestingly, our analysis only revealed copy number variations between the two species of tardigrades in genes related to DNA repair mechanisms, particularly in genome maintenance during replication, double-strand break (DSB) repair, and nucleotide excision repair (NER) pathways (Table 2; Supplementary Table 2). However, no relevant copy number alterations were found in genes related to telomere maintenance, stress response or oxygen homeostasis when comparing the genomes of R. varieornatus and H. dujardini. Moreover, our analysis of DNA repair pathways in tardigrades and their comparison with reported data on human sequences led us to identify a series of residue changes that are exclusive of R. varieornatus and/or H. dujardini (Supplementary Table 3).
In this study, we focused on the description of copy number variations and residue changes exclusive of the extreme tolerant R. varieornatus that lay in active sites or DNA binding sites, and involve genes important for homologous recombination, base excision repair, nucleotide excision repair, non-homologous end-joining, translesion synthesis, DNA repair during replication (Table 2), and for telomere dynamics.
Telomere dynamics in R. varieornatus and H. dujardini
Telomeres have been widely studied in all Arthropoda, being their ancestral sequence (TTAGG)n common to hexapods, crustaceans, myriapods, pycnogonids and most chelicerates, but not to spiders27. Nonetheless, such repeat sequence is absent in Tardigrada and Onychophora, which are closely related to Arthropoda. Thus, Onychophora present the vertebrate motif (TTAGGG)n, while tardigrades do not exhibit this telomere sequence either27. Further analysis of repeat sequences in the genome of H. dujardini revealed the presence of (GATGGGTTTT)n repeats, which were exclusively found at 9 scaffold ends and are thought to correspond to telomeric sequences11 located in its 5 pairs of chromosomes28. Moreover, tardigrades and most arthropods lack the TERT motif CP, with the exceptions of hymenopterans and some centipedes29. This motif, together with the T motif, forms an extended pocket (T-CP pocket) on the surface of the protein implicated in RNA recognition and binding30. Remarkably, telomere elongation in D. melanogaster is carried out by three specialized retrotransposable elements (HeT-A, TART and Tahre)31, while no ortholog for the human gene TERT has been reported. In addition, fruit fly telomeres are capped by the complex terminin, functionally but not structurally analogous to shelterin, which includes the proteins HOAP, HipHop, Moi and Ver32,33 (Fig. 1a). These data indicate that telomere elongation and maintenance are carried out through different mechanisms in this species in contrast to other members of the Metazoa group.
In this work, we manually annotated several genes that encode proteins belonging to the telomerase, shelterin and CST complexes in tardigrades (Fig. 1a). Except for TPP1, none of the other components from the shelterin (TERF1, TERF2, RAP1, POT1, and TINF2) and CST (CTC1, STN1 and TEN1) complexes were identified (Fig. 1a, CST complex not shown). Interestingly, we found in tardigrades a bona fide TERT ortholog, together with copies encoding all the elements of the telomerase complex, namely NHP2, NOP10, DKC1 and GAR1 (the latter being duplicated in H. dujardini) (Fig. 1a). Remarkably, two residue changes in TERT protein - p.L866Y and p.V867M - were found to be exclusively present in H. dujardini and R. varieornatus (Fig. 1b). Both residues are part of a tetrapeptide that includes a catalytically essential aspartate dyad (residues D868 and D869)34. These residues have been studied based on the previous discovery of the function of Y183 and M184, cognate amino acids to human TERT L866 and V867 in HIV-1 reverse transcriptase (Fig. 1b), which play important roles in processing, fidelity, enzymatic activity, dNTP utilization and nucleoside analogue inhibitor resistance35. These functional studies in human TERT have shown that the first variant alone (p.L866Y) results in a moderate reduction in telomerase activity, but produces no changes in repeat extension rate or in nucleotide incorporation fidelity34. The second variant (p.V867M) causes a 75% reduction in telomerase activity, 50% reduction in repeat extension rate, and 5.2-fold increase in nucleotide incorporation fidelity34. However, when both variants are present, they result in a slight reduction in telomerase activity and 13.5-fold increase in nucleotide incorporation fidelity34. This finding suggests that telomere dynamics in tardigrades may display reduced telomerase activity but also enhanced replication fidelity to prevent genomic instability caused by defects in telomere maintenance20.
Alterations in genes involved in DNA repair and genome maintenance during replication in tardigrades
DNA ligation is essential for replication and repair, and genetic deficiencies in human DNA ligases have been associated with clinical syndromes characterized by radiation sensitivity and defects in DNA repair during replication through nonhomologous end joining (NHEJ)36. In mammals, this functional role is carried out by a protein family encoded by three genes (LIG1, LIG3 and LIG4), all of them also present in D. melanogaster. While both tardigrade species seem to have one copy of LIG1 and none of LIG3, we found two copies of LIG4 in the genome of R. varieornatus (called LIG4_1 and LIG4_2), while only one full copy and what could be one exon of another copy were detected in the genome of H. dujardini. The presence of this second LIG4 copy in H. dujardini could not be verified by RNA-Seq nor Sanger sequencing due to the shortness of its contig (Supplementary Table 4), even though a putative expansion of LIG4 in H. dujardini has been previously suggested11. Nevertheless, supporting data in this regard are not available in public repositories of genomic data11. Importantly, patients with null mutations in LIG4 show increased sensitivity to ionizing radiation, as well as immunodeficiency, growth failure, and microcephaly37. In mice, Lig4 deficiency causes embryonic lethality due to a defective p53-dependent response to unrepaired DNA damage, as well as neuronal apoptosis and arrested lymphogenesis38. Moreover, mice with a hypomorphic mutation in Lig4 show high levels of DNA DSBs during embryonic development and a deficient DSB repair response39. Accordingly, LIG4 mediates Wnt/β-catenin signaling activation during radiation-induced intestinal regeneration and blocking LIG4 sensitizes colorectal cancer cells to radiation40. Since the second copy of H. dujardini is not experimentally supported, it is plausible that the exclusive presence of two copies of LIG4 in R. varieornatus might contribute to its enhanced resistance to DNA damage.
Moreover, we found several remarkable residue changes in CHEK1 (Supplementary Table 3), which codes for the protein kinase CHK1 involved in DNA damage response (DDR), cell cycle arrest, and homologous recombination (HR)41. Among these CHEK1 variants, we focused our attention on p.F93Y, exclusive of R. varieornatus (Fig. 2a), which affects an active site that functions as an allosteric inhibitor binding site and as a polypeptide substrate binding site42. To explore the putative functional relevance of this change, we generated a homology model of this protein in R. varieornatus (Fig. 2b). This model revealed that position 93 is located at the surface of a pocket in which allosteric inhibitors can be fitted, and showed the potential of the residue Y93 to form an H-bond with a synthetic allosteric inhibitor (Fig. 2b)42. This amino acidic change might influence the allosteric regulation of CHEK1 in R. varieornatus in comparison to H. dujardini. This regulatory mechanism may be important for its function, since CHK1 is involved in DNA damage response (DDR), cell cycle arrest, and homologous recombination (HR)41.
We also found an alteration (p.S132G) in the polymerase POLK exclusive of R. varieornatus (Fig. 3a), together with other residue changes shared with H. dujardini (Supplementary Table 3). The p.S132G variant affects a residue involved in DNA binding43. POLK is an error-prone DNA polymerase specifically involved in translesion synthesis during DNA replication, which preferentially incorporates adenine residues opposite to 8-oxoguanine lesions. These lesions frequently appear as a result of ionizing radiation, therefore producing missense mutations and frameshifts43,44. POLK appears to be absent in all arthropods. Its prokaryotic ortholog, DNA polymerase IV45, is also involved in repair of 8-oxoguanine lesions, but incorporates cytosine instead of adenine opposite to 8-oxoguanine with high efficiency, thus avoiding potential mutations46. Notably, prokaryotic DNA polymerase IV also presents glycine instead of serine in residue 132 (Fig. 3a), which suggests that the presence of glycine may contribute to incorporating the right nucleotide during repair of 8-oxoguanine lesions, resulting in higher fidelity and decreasing the occurrence of point mutations. The homology model of this protein in R. varieornatus suggests that, although the position 132 is not strictly close to the 8-oxoguanine lesion, it contributes to creating a more acute beta turn (Fig. 3b). Finally, REV1 - another protein involved in translesion synthesis47 - presents a variant exclusive of R. varieornatus affecting a DNA binding site (p.A509S)48,49. Additionally, R. varieornatus REV1 presents other changes in DNA binding sites that are also found in H. dujardini (Supplementary Table 3).
Finally, the gene MGMT, which encodes a methyltransferase involved in repairing the naturally occurring mutations O6-methylguanine and O4-methylthymine during replication50, is present in H. dujardini but the corresponding ortholog in R. varieornatus had not been previously identified in manual and automatic annotations. However, we could confirm the presence of MGMT when performing PCR on the genome of R. varieornatus using oligonucleotides based on the corresponding MGMT sequence of H. dujardini (Supplementary Table 4). Accordingly, its apparent absence in R. varieornatus genome is likely due to errors in the currently available genome assembly for this tardigrade.
Expansion of genes involved in double-strand break repair in tardigrades
DSBs are particularly damaging alterations, since they can lead to chromosome rearrangements and losses. These genomic lesions can be repaired through three mechanisms: NHEJ, HR and microhomology-mediated end joining (MMEJ)51. We confirmed that the human MRE11 ortholog, involved in NHEJ and HR52, is at least quadrupled in R. varieornatus, while H. dujardini displays one copy (Supplementary Table 4), as it has previously been reported9,11. The remarkable expansion of this gene may be responsible for an enhanced ability to repair DNA damage53. Moreover, knockdown of MRE11 impaired DSB repair in HeLa and CNE2 cells54, and upregulation of this protein in cancer cells following ionizing radiation promoted DNA repair54. Altogether, these data suggest an important role of MRE11 ortholog in R. varieornatus in promoting DNA repair after exposure to ionizing radiation.
We also confirmed the previous finding that the RAD51 protein family, involved in DSB repair through HR55, is expanded in R. varieornatus9. However, according to our data, we propose that one of the four copies annotated in this tardigrade’s genome by Hashimoto et al. actually corresponds to the XRCC2 ortholog, as assessed by performing blast of these sequences (deposited in the NCBI database) against the human genome. Therefore, according to our annotation, the genome of R. varieornatus contains three copies of RAD51. We independently found the presence of the other three copies in both tardigrades. Expansion of the DNA repair endonuclease XPF (encoded by the gene ERCC4), also involved in HR56, was reported in H. dujardini, since five copies of this gene were found in its genome11. However, only three sequences out of these five could be found in the NCBI database, two of which belong to very small polypeptides (<100 aa); and only one is available at Ensembl Tardigrades11. In turn, manual annotation of this gene revealed two copies of ERCC4 in this species (named ERCC4_1 and ERCC4_2), while only one copy was found in R. varieornatus. This duplication could be verified by RNA-Seq, but not using Sanger sequencing due to the high similarity between both copies, and the presence of repetitive sequences (Supplementary Table 4). Finally, and similarly to the case of MGMT, one copy of the gene XRCC3, also involved in HR, could be found in the genome of H. dujardini. Although this gene seemed to be absent in the genome of R. varieornatus, we detected it by PCR using oligonucleotides designed for H. dujardini (Supplementary Table 4).
Changes in genes related to base excision repair in R. varieornatus
Among all genes involved in base excision repair (BER) analysed in R. varieornatus and H. dujardini, we found a variant in an active site and UGI (uracil-DNA glycosylase inhibitor protein) interface site (p.P177R) in the protein encoded by UNG that is exclusive of R. varieornatus57 (Fig. 4a). This protein is a DNA glycosylase that excises uracil residues from DNA when misincorporation of uracil occurs during DNA replication or due to deamination of cytosine58. The model predicts that Arg 177 fits the minor groove of the DNA molecule, very close to the everted base (Fig. 4b). This mode of interaction has been described previously in the context of the nucleosome, and it was found to be independent of the DNA sequence59, which suggests that this variant might contribute to the association of UNG to substrate DNA. In this regard, another tardigrade-specific arginine at position 256 (Fig. 4a) interacts with a phosphate group at the other side of the everted base. However, given the proximity of Arg177 to the substrate base, this model cannot rule out the possibility that this residue might also play a role in base eversion, as proposed in similar contexts for other enzymes60.
Nucleotide excision repair in R. varieornatus
Oxidative DNA damage is considered as a leading cause of both neurodegeneration and cancer development as illustrated by syndromes that result from NER defects, such as Xeroderma pigmentosum (XP) and Cockayne syndrome (CS)61,62. Among all the genes involved in NER, XPC appears to be duplicated in R. varieornatus (with copies we have named XPC_1 and XPC_2) but not in H. dujardini (Supplementary Table 4). This protein is involved in repair of damage caused by UV light, since mutations in the gene encoding this protein in humans lead to XP61, and Xpc knockout mice show an increased susceptibility to UVB induced squamous cell carcinomas63. Therefore, this duplication in the XPC ortholog in R. varieornatus may also contribute to the enhanced tolerance to radiation in this species by improving its NER response pathway.
Summary
In this manuscript, we describe several gene expansions of pivotal elements in DNA repair pathways observed in the genomes of R. varieornatus and H. dujardini through manual annotation, including previously described expansions, such as XPC, LIG4, ERCC4 and MRE119,11. Manual genomic comparative analyses also revealed residue changes in key elements in DNA repair pathways that in the corresponding human orthologs are known to cause an effect in the function of the protein (Supplementary Table 3), among which we highlight the ones exclusively found in R. varieornatus in the genes TERT, CHEK1, POLK and UNG. However, considering the phylogenetic distance between tardigrades and humans, in most cases it is difficult to define the consequences of such variants in tardigrade proteins, and further experimental work is required to raise definitive conclusions in this regard. Nonetheless, these findings show that combining both manual and automatic annotation approaches is an advantageous strategy to better determinate the precise number of gene copies and to find residue changes when analyzing a genome de novo.
In short, all the changes we observed in R. varieornatus suggest an enhanced ability to maintain genomic stability, which may explain its resistance to extreme conditions, as well as its longer lifespan in comparison to H. dujardini. Additionally, the recent finding of a Dsup homologue in H. dujardini17 reinforces our proposal that specific features in DNA repair genes are important elements in the extraordinary resistance shown by this limno-terrestrial tardigrade species.
Data availability
The manually annotated dataset of genes and proteins generated and analyzed during the current study supporting the conclusions of this article are available in a public repository in GitHub(https://github.com/EreboPSilva/rvar.hduj.prots).
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Acknowledgements
We thank Alicia R. Folgueras and J.M. Freije for helpful comments and advice, and T. Kunieda for providing valuable biological materials. This work was supported by grants from European Research Council (DeAge, ERC Advanced Grant), Ministerio de Economía y Competitividad, Instituto de Salud Carlos III (Ciberonc) and Progeria Research Foundation. The Instituto Universitario de Oncología is supported by Fundación Bancaria Caja de Ahorros de Asturias.
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D.C. performed manual annotation of genomes, data interpretation and preparation of the manuscript. J.G.P.S. prepared the analyzed genomes for their manual annotation and performed bioinformatic analyses. V.Q. performed protein modelling and supervised data interpretation. C.L.O. supervised research and project planning, data interpretation and preparation of the manuscript.
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Carrero, D., Pérez-Silva, J.G., Quesada, V. et al. Differential mechanisms of tolerance to extreme environmental conditions in tardigrades. Sci Rep 9, 14938 (2019). https://doi.org/10.1038/s41598-019-51471-8
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DOI: https://doi.org/10.1038/s41598-019-51471-8
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