Telomeric RNA (TERRA) increases in response to spaceflight and high-altitude climbing

Telomeres, the protective caps at chromosome ends, are crucial for genome stability. They face two main challenges: the end replication problem (inability to fully replicate linear DNA) and the end protection problem (avoiding inappropriate DNA damage responses). Mammalian telomeres consist of repetitive G-rich sequences, with lengths varying between individuals and influenced by factors like age and oxidative stress. Telomerase, a reverse transcriptase, adds telomeric repeats, but its activity is low in most somatic cells, leading to telomere shortening with age. A significant minority of cancers, however, maintain telomere length through the Alternative Lengthening of Telomeres (ALT) pathway, a telomerase-independent mechanism involving recombination. The discovery of Telomere Repeat-containing RNA (TERRA), a long noncoding RNA transcribed from telomeres, has opened new avenues of research into telomere maintenance. TERRA's length varies considerably, and it's transcribed from subtelomeric regions by RNA Pol II, predominantly using the C-rich strand as a template. TERRA's primary roles include contributing to telomere end-capping and regulating telomere length. It interacts with shelterin proteins and RNA-binding proteins, influencing genome stability. In telomerase-positive cells, most TERRA is chromatin-associated and non-polyadenylated, whereas free TERRA is polyadenylated. TERRA can form R-loops with telomeric DNA, creating replication stress and DNA damage. The interaction between TERRA and telomeric DNA can also produce TERRA:telomeric DNA hybrids, which play crucial roles in ALT cells, particularly in the absence of the ATRX/DAXX complex. These hybrids contribute to telomere protection and recombination-mediated elongation. Previous research utilizing site-specific endonucleases to create telomeric double-strand breaks (DSBs) highlighted the potential role of TERRA in repairing these breaks. This study expands upon this by examining TERRA's role in the DNA damage response (DDR) following ionizing radiation and specifically at telomeric DSBs, using samples from astronauts, high-altitude climbers, and cellular models.

Existing literature extensively documents the crucial role of telomeres in maintaining genome stability and their association with aging and cancer. Studies have shown that telomere shortening is a hallmark of aging and is linked to various age-related diseases. The discovery of TERRA has significantly advanced our understanding of telomere biology, revealing its involvement in telomere length regulation and maintenance. Research has demonstrated TERRA's ability to interact with telomeric DNA, forming R-loops and hybrids, and its function in the ALT pathway in cancer cells. The role of TERRA in the DNA damage response, particularly in the context of telomeric double-strand breaks, is a relatively newer area of investigation with studies beginning to show that TERRA may protect telomeres and facilitate repair mechanisms. The effect of spaceflight and high-altitude environments on telomere length and the potential activation of ALT-like mechanisms has also been studied, though the mechanistic details require further investigation. This study builds on these previous findings to examine the relationship between TERRA, telomeric DNA damage, and exposure to extreme environments such as spaceflight and high altitude.

This study used a multi-pronged approach combining in vivo and in vitro analyses. For in vivo analysis, RNA sequencing (RNA-seq) data from astronauts on long-duration (NASA Twins Study) and short-duration (SpaceX Inspiration4) spaceflights were analyzed for TERRA transcript abundance. Similarly, RNA-seq data from high-altitude climbers ascending Mount Everest were examined. Simulated microgravity experiments using rotating wall vessels with PBMCs from healthy donors were conducted to isolate the effect of microgravity on TERRA expression. In vitro experiments utilized U2OS (ALT) cells exposed to ionizing radiation (IR) to study TERRA's response to radiation-induced DNA damage. The effect of transcription inhibition on TERRA induction was assessed using Actinomycin-D. To specifically target telomeric DSBs, the TRAS1-EN-TRF1 (EN-T) targeting system was employed in U2OS cells, enabling the investigation of TERRA's response to telomere-specific damage. RNA fluorescence in situ hybridization (RNA-FISH) was used to visualize and quantify TERRA foci, and the co-localization of TERRA with other markers (FLAG, γ-H2AX, TRF2) was analyzed to assess TERRA's presence at damaged telomeres. To distinguish between bound and unbound TERRA, cells were treated with RNaseA and RNaseH. Cell cycle analysis using a FUCCI-Geminin expressing U2OS cell line helped determine TERRA's behavior throughout the cell cycle in the context of telomeric DSBs. Statistical analyses included Mann-Whitney U tests, Benjamini-Hochberg correction, Pearson's correlation, and ANOVA with post-hoc tests.

RNA-seq data from both long- and short-duration spaceflights showed a significant increase in TERRA transcript abundance compared to pre-flight baselines. Similarly, high-altitude climbers exhibited elevated TERRA levels at high altitudes, which returned to baseline after descent. In contrast, simulated microgravity did not induce a significant increase in TERRA. In vitro IR exposure in U2OS cells led to a significant increase in TERRA foci, which was transcription-dependent, as indicated by Actinomycin-D treatment. The EN-T system, which induces telomere-specific DSBs, demonstrated a direct visualization of hybridized TERRA at these DSB sites. Analysis showed a significant increase in TERRA co-localization with telomeric DSBs and a shift in TERRA distribution towards a higher proportion of bound TERRA in cells with DSBs. Cell cycle analysis revealed a G2-phase accumulation of TERRA in cells with telomeric DSBs, suggesting a role in recombination-mediated repair. The increase in TERRA was not only evident in cells exposed to IR but also in cells experiencing the cellular stress of transfection.

This study provides compelling evidence for a telomere-specific DDR triggered by chronic oxidative stress and telomeric damage, which is observed in the space radiation environment and at high altitudes. The increase in TERRA transcription and its subsequent recruitment to telomeric DSBs suggest a protective role, preventing further resection and facilitating repair through RNA-templated homologous recombination. The accumulation of TERRA in G2 further supports its role in recombination-mediated telomere elongation. The observed increase in TERRA in response to both radiation and general cellular stress suggests a broader response to various stressors impacting telomere integrity. The fact that simulated microgravity did not induce similar increases in TERRA indicates that the observed effects are likely driven primarily by radiation exposure and/or oxidative stress.

This research reveals a novel telomere-specific DNA damage response mechanism where TERRA is upregulated in response to telomeric damage caused by chronic oxidative stress and radiation. This response involves the recruitment of TERRA to damaged telomeres, forming protective hybrids, and facilitating repair through homologous recombination, potentially contributing to transient ALT activation. These findings have broad implications for understanding the effects of spaceflight, high-altitude exposure, and chronic oxidative stress on telomere maintenance, and potential therapeutic targeting of TERRA in ALT-positive cancers.

The study primarily focused on U2OS (ALT) cells, which may not fully represent the behavior of normal human cells. The simulated microgravity model may not perfectly mimic the complex environment of space. The sample size for in vivo studies, particularly those involving astronauts, is limited. The study does not provide a fully comprehensive mechanistic analysis and future research focusing on specific molecular interactions in this process are warranted.