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

The study investigates whether telomeric RNA (TERRA) participates in DNA damage responses relevant to spaceflight and high-altitude exposure, environments characterized by elevated radiation and chronic oxidative stress. Telomeres are repetitive G-rich DNA-protein structures that protect chromosome ends but are susceptible to replication and end-protection problems and to oxidative damage. TERRA is transcribed from subtelomeric regions and can interact with telomeric DNA and binding proteins, influencing telomere maintenance, genome stability, and recombination, especially in ALT cells. Prior human studies showed paradoxical telomere elongation during spaceflight and high-altitude climbing along with ALT-like signatures, suggesting stress-induced telomere dynamics. The central hypothesis is that TERRA is upregulated in response to radiation-induced and telomere-specific DNA double-strand breaks, accumulates at damaged telomeres, forms protective DNA:RNA hybrids, and may facilitate recombination-mediated repair, contributing to observed telomere length dynamics in vivo.

The paper summarizes extensive prior work on telomere biology, including shelterin components (TRF1/TRF2), telomerase activity, and ALT mechanisms. ALT tumors exhibit heterogeneous telomere lengths, increased telomere sister chromatid exchange, APBs, and extrachromosomal telomeric repeats. TERRA, historically considered an lncRNA, is transcribed by RNA Pol II, accumulates at short telomeres, and can form R-loops or two-stranded TERRA:telomeric DNA hybrids that promote homology-directed repair, particularly in ALT cells where ATRX/DAXX dysfunction leads to elevated TERRA. RNaseH1 overexpression in ALT cells shortens telomeres, implicating TERRA:DNA hybrids in telomere protection and elongation. Telomeres are prone to oxidative damage and may be refractory to repair; telomere-specific nucleases (e.g., EN-T) have been used to interrogate DSB repair at telomeres. Previous human studies showed telomere elongation and ALT-like activity during spaceflight and high-altitude climbing, and stressors including radiation, mitochondrial dysregulation, and inflammation correlate with telomere dynamics. Prior in vitro reports showed TERRA upregulation following DNA-damaging agents (bleomycin, etoposide) and oxidative stress, suggesting crosstalk between cellular stress and TERRA induction.

Human in vivo studies: RNA-Seq datasets were analyzed from astronauts in two cohorts: NASA Twins Study (One-Year mission, bulk poly(A)+ RNA-Seq) and SpaceX Inspiration4 (3-day mission, single-nucleus multiome and direct RNA sequencing). Telomeric TERRA motifs (5–7-mers) including UUAGGG and highly correlated variants were quantified using jellyfish k-mer counting, normalized, and compared across timepoints categorized as ground, space effect (in-flight or immediate post-flight), and recovery. Mann–Whitney U tests with Benjamini–Hochberg FDR correction assessed differential motif abundance. High-altitude Mt. Everest climbers (two males) were sampled longitudinally (low altitude pre-ascent, high altitude ascent at Everest Base Camp and Camp II, and low altitude descent) with analogous k-mer analyses. Simulated microgravity experiments used PBMCs from two healthy donors cultured for 25 hours in rotating wall vessels versus 1G controls; single-cell RNA-Seq libraries were prepared and analyzed similarly for TERRA motif abundance. In vitro radiation experiments: U2OS ALT cells were exposed to 2 Gy 137Cs γ-rays or sham-irradiated; four hours later, TERRA foci were quantified by RNA-FISH with 3D imaging. RNaseA and RNaseH treatments distinguished free versus hybridized TERRA. To test transcription dependence, global transcription was inhibited with actinomycin D (10 μg/ml) for 3 or 8 hours prior to irradiation; ddPCR of housekeeping genes (GAPDH, 18S) confirmed transcriptional inhibition kinetics. Telomere-specific DSB targeting: Telomeric DSBs were induced by transient transfection with TRAS1-EN fused to TRF1 (EN-T), with TRF1-only and empty vector controls. Transfection efficiency was assessed via FLAG immunostaining; only cells with ≥20 FLAG foci were analyzed. Telomeric DSBs were validated by triple co-localization of FLAG, γ-H2AX, and telomere FISH signals. TERRA recruitment to damaged telomeres was quantified by triple co-localization of FLAG, TRF2 (telomeres), and TERRA RNA-FISH, with and without RNaseA+H treatment. Distribution of bound (hybridized) versus free TERRA was estimated by comparing total TERRA foci to hybridized TERRA foci. Cell cycle analyses: A U2OS line stably expressing FUCCI/Geminin-GFP identified G2 cells; EdU pulse-labeling identified S-phase; unlabeled cells were G1. TERRA foci were quantified across cell-cycle phases in transfected and control populations. Imaging used Zeiss Axio Imager.Z2 with 3D z-stacks; quantification used Fiji/ImageJ and CellProfiler. Statistical analyses employed ANOVA with multiple-comparisons tests, and nonparametric tests for sequencing data with FDR correction.

• Spaceflight: TERRA motif abundance (UUAGGG and highly correlating variants) significantly increased during spaceflight and immediately post-flight compared to ground and recovery samples in both cohorts. Mann–Whitney U tests (FDR-adjusted p < 0.05) showed concerted enrichment; top motif correlations reached Pearson r = 0.99 (Twins Study) and r = 0.87–0.94 (Inspiration4). Motif levels returned to baseline during recovery.
• Simulated microgravity: No significant increase in TERRA motif abundance after 25 h simulated microgravity in PBMCs (Mann–Whitney U p = 0.21), indicating microgravity alone did not induce TERRA.
• High-altitude climbing: Climbers exhibited significant TERRA motif enrichment at high altitudes (Everest Base Camp, Camp II) versus low altitudes (Denver, Kathmandu), with levels returning to baseline after descent (FDR-adjusted p < 0.05), paralleling prior observations of telomere elongation and ALT-like activity during ascent.
• In vitro IR exposure: U2OS cells exposed to 2 Gy γ-rays showed a significant increase in mean TERRA foci per nucleus relative to controls (p ≤ 0.0001). RNaseA+H treatment markedly reduced TERRA foci, validating RNA identity and DNA:RNA hybrids; RNaseA alone reduced but did not eliminate signals. Pre-treatment with actinomycin D (3 or 8 h) abrogated the IR-induced increase in TERRA foci, demonstrating transcription-dependent induction.
• Telomere-specific DSBs: EN-T and TRF1-only transfections induced telomeric DSBs verified by FLAG–γ-H2AX–telomere triple co-localization (>80% telomeres damaged in transfected cells). TERRA was directly observed at telomeric DSB sites via FLAG–TRF2–TERRA triple co-localization, significantly increasing at damaged telomeres (p < 0.0001) while decreasing at undamaged telomeres.
• Redistribution of TERRA: In EN-T and TRF1-only conditions, approximately 90% of total TERRA became telomere-bound (hybridized) with only ~10% free, versus control populations with ~30% bound and ~70% free, indicating rapid recruitment of free TERRA to DSBs and formation of protective DNA:RNA hybrids.
• Cell-cycle dependence: Transfected populations accumulated in G2-phase consistent with a DNA damage-induced G2 block. TERRA foci were significantly elevated in G2 versus G1 in transfected cells (p < 0.0001), supporting a role for TERRA in recombination-mediated repair during G2.

The findings demonstrate that TERRA is part of a telomere-specific DNA damage response relevant to the space radiation environment and high-altitude exposure. Increased TERRA transcription was consistently observed in vivo during spaceflight and at high altitude, but not under simulated microgravity, implicating radiation and oxidative stress rather than gravity changes as the main drivers. In vitro, TERRA upregulation following γ-irradiation was transcription-dependent and accompanied by recruitment and retention of TERRA at telomeric DSBs, forming TERRA:telomeric DNA hybrids that likely protect resected C-rich overhangs and promote RNA-templated, homologous recombination-based repair. The redistribution of TERRA from free to bound states and accumulation in G2 support a mechanistic model where TERRA facilitates recombination-mediated telomere elongation after damage. These data align with prior observations of ALT-like telomere dynamics during spaceflight and high-altitude ascent and suggest that persistent telomeric damage from chronic oxidative stress and radiation can transiently activate ALT-like processes in normal cells. The potential dose-rate dependence noted between missions further motivates investigation of TERRA as a biomarker and mediator of telomere responses to space radiation. Clinically, modulating TERRA or its hybrids could be leveraged to improve outcomes in ALT-positive cancers and mitigate risks associated with chronic telomeric damage in aging and inflammatory states.

This study provides mechanistic evidence that TERRA increases during spaceflight and high-altitude exposure and is specifically recruited to telomeric DSBs, forming protective DNA:RNA hybrids that support recombination-based repair in G2. The integration of human in vivo datasets with targeted cellular models shows that radiation and oxidative stress, rather than microgravity, drive TERRA induction. These insights help explain observed telomere elongation and ALT-like phenotypes in extreme environments and suggest avenues for aerospace precision medicine. Future research should quantify TERRA dose–response relationships to space radiation, dissect regulatory controls that restrain ALT-like activity in normal cells, evaluate TERRA’s roles in telomere mobility and repair complex recruitment, and explore therapeutic targeting of TERRA-mediated repair in ALT-positive tumors.

• Simulated microgravity platforms are terrestrial analogs that may not precisely replicate space microgravity, potentially limiting direct comparisons.
• Human cohorts were limited (single astronaut in Twins Study, four in Inspiration4, and two climbers), constraining generalizability and statistical power.
• Inspiration4 data exhibited lower motif correlation coefficients than Twins Study, potentially due to RNA degradation or short mission duration, indicating variability in data quality and exposure duration.
• In vitro mechanistic studies relied on U2OS ALT cells and transient transfection, which introduces cellular stress and may not reflect telomerase-positive normal cells. Empty vector controls showed stress-related TERRA increases, highlighting possible confounding effects.