Tardigrades dramatically upregulate DNA repair pathway genes in response to ionizing radiation

Courtney M. Clark-Hachtel,Jonathan D. Hibshman, Tristan De Buysscher,Bob Goldstein

Preprint posted on 7 September 2023

What doesn’t kill you makes you stronger: @ArthropodQueen and team characterize a novel mechanism of ionizing radiation tolerance in the tardigrade H. exemplaris

Selected by Isabella Cisneros


Colloquially known as water bears or moss piglets, tardigrades are fascinating invertebrate organisms with the ability to survive a variety of extreme conditions, including extreme temperature, desiccation, and even the harsh environment of outer space [1]. When exposed to these conditions, tardigrades can enter a state called cryptobiosis, in which their metabolism comes to a reversible standstill until more suitable conditions are reached [1,2]. This aspect of tardigrade biology has become a subject of great interest and is increasingly being studied, with genomes for the tardigrade speciesRamazzottius varieornatusandHypsibius exemplarisalready available [3,4].

Researchers have already begun to characterize the mechanisms underlying cryptobiosis, particularly in the case of DNA repair. Previous studies in the tardigrade speciesR. varieornatusfound a DNA damage suppressing protein—termedDsup——对电离辐射的产生了耐药性) by binding to nucleosomes and protecting DNA from hydroxyl radicals [3,5]. However, little else is known about the mechanisms that tardigrades use to survive DNA damage resulting from extreme conditions. In this preprint, the authors sought to understand how the tardigradeH. exemplarissurvives the DNA damage caused by ionizing radiation. Using sequencing and molecular techniques, the authors describe a novel mechanism underlying IR tolerance in tardigrades.

Main Findings:

Messenger RNA sequencingreveals significant upregulation of DNA repair genes following exposure to ionizing radiation

After confirming that IR induces DNA damage inH. exemplarisand that the tardigrade is able to repair its damaged cells post-exposure, the authors set out to understand the mechanisms underlying this repair. To do so, the authors performed messenger RNA sequencing (mRNA-seq) on tardigrades post-exposure to either 100, 500, or 2180 Gray (Gy) doses of IR. These doses range from a level with proven survival and reproduction post-exposure to an LD50 dose (a dose of IR at which 50% of humans would die).

Analysis of differential expression showed a robust response fromH. exemplaris, with about half of the most enriched transcripts encoding proteins found in DNA repair pathways. Additionally, these transcripts were found to be involved in base excision repair (BER) and non-homologous end joining (NHEJ), two important DNA repair processes that lend themselves to repairing the damage caused by IR. The authors also analyzed transcript enrichment for genes associated with other reparative processes such as mismatch repair (MMR), nucleotide excision repair (NER), homologous recombination (HR) and theta-mediated end joining (TMEJ). They found transcripts associated with HR as well as TMEJ, revealing specificity in the tardigrade transcriptional response to IR damage.

DNA repair transcripts identified through sequencing are expressed throughout the tardigrade but are also enriched in specific tissues

Having characterized the transcriptional response to IR, the authors next asked whether this response is tissue-specific or an overall response. They performedin situhybridization on a few of the enriched DNA repair transcripts identified through mRNA-Seq. Following IR exposure, all of the transcripts observed were enriched in nearly all tissues, validating their sequencing results. However, some transcripts were particularly enriched in secretory tissues, such as the salivary glands and the hindgut, while nearly all were enriched in storage cells called coelomocytes. Given that secretory tissues are believed to be active in transcription and translation, the authors attributed this enrichment to the fact that transcriptionally active tissues are more sensitive to damage caused by IR.

Figure 1: In situhybridization of enriched DNA repair transcripts. (A-D) Detection of the DNA repair transcriptsLIG1,PARP3,XRCC5, andRAD51under two treatment conditions: no exposure to ionizing radiation or exposure to 100 Gy. Arrows indicate salivary glands, arrowheads indicate claw glands, and dashed circles correspond to the hindgut. (E) Schematic of an adult tardigrade showing the regions highlighted above as well as other major anatomical features.

Heterologous expression of DNA repair transcripts in bacteria confers resistance to radiation—but degree of expression matters

The authors next chose to test whether the DNA repair genes that showed increased expression inH. exemplarisconfer protection against IR. They expressed these genes heterologously inE. coliand exposed colonies to 2,180 Gy. They then observed these colonies to see how their survival compared to negative controls as well as a positive control vector expressingDsup, a previously characterized tardigrade gene shown to improve radiation survival in human cells.

All but one of the introduced genes that increased survival ofE. coli后殖民地红外编码proteins in the BER pathway. For some of these genes, expression resulted in the same levels of protection as that of theDsupprotectant. The authors were curious to see if high levels of expression of these genes were necessary for the survival of tardigrades after IR exposure, which they tested by using RNA interference (RNAi). The geneXRCC5was selected as it was the most significantly enriched transcript following IR exposure. They found that tardigrades injected with dsRNA targeting this gene show more lethality compared to controls, indicating that dramatic upregulation of DNA repair genes plays a significant role in the overall response to IR.

Why I Chose This Preprint:

I find the novel mechanism of tardigrade IR tolerance characterized in this preprint to be absolutely fascinating, especially given both the current gap of knowledge regarding tolerance mechanisms as well as the fact that this mechanism differs from the previously characterizedDsup. While tardigrades are not a commonly used model organism, there is quite a lot to be learned from them, particularly when it comes to survival under extreme conditions. Further characterization of tardigrade tolerance could have significant applications in a variety of contexts, from climate change to future space exploration. This study marks an exciting step forward in the characterization of tolerance responses and I am excited to see how this research continues to develop.

Questions For The Authors:

  1. How did you decide upon the 24hr post-exposure timepoint for your experiments? Did you consider incorporating later timepoints for sequencing and/orin situexperiments?
  2. In the discussion, you mention the possibility of synergy between multiple protective mechanisms. However, heterologous expression ofexemplaris Dsupdoes not confer the protection given byR. varieornatus Dsup. How likely do you think the occurrence of synergy is, and what could be the benefit of not having it?
  3. While it is possible that strong IR tolerance developed from cross-tolerance for another threat, how do we account for the significant upregulation of DNA repair genes shown by the tardigrades? Is it possible that the upregulation of DNA genes seen in this study is tied to the extent of DNA damage induced by ionizing radiation (as compared to the damage caused by desiccation, for example)?


[1] Møbjerg, N. & Cardoso Neves, R. New insights into survival strategies of tardigrades.Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 254:110890 (2021).

[2] Halberg, K. A., Jørgensen A., Møbjerg, N. Desiccation Tolerance in the TardigradeRichtersius coroniferRelies on Muscle Mediated Structural Reorganization.PLoS ONE, 8(12): e85091 (2013).

[3] Hashimoto, T., Horikawa, D., Saito, al.Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein.Nat Commun7, 12808 (2016).

[4] Koutsovoulous, G., Kumar, S., Laet, D. R. et al. No evidence for extensive horizontal gene transfer in the genome of the tardigrade Hypsibius dujardini.Proc Natl Acad Sci USA, 113(18):5053-5058 (2016).

[5] Chavez, C., Cruz-Becerra, G., Fei, J., Kassavetis, G. A., Kadonaga, J. T. The tardigrade damage suppressor protein binds to nucleosomes and protects DNA from hydroxyl radicals.eLife8:e47682 (2019).

Tags:dna repair,ionizing radiation,rna sequencing,tardigrade,tolerance

Posted on: 18 October 2023 , updated on: 19 October 2023


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