DHX30 is a replication stress-responsive R-loop helicase that safeguards genome stability

Nedelcu, E. R.; Yeo, N. C.; Huber, H.; Prusty, A. B.; Truong, T. Q.; Dang, N. D. P.; Harder, S.; Lederbauer, J.; Lessel, I.; Ripin, N.; Kiel, C.; Fischer, U.; Kreienkamp, H.-J.; Lessel, D.

doi:10.64898/2026.10.25.761626

bioRxiv · 10.64898/2026.10.25.761626

DHX30 is a replication stress-responsive R-loop helicase that safeguards genome stability

Nedelcu, E. R.·Yeo, N. C.·Huber, H.·Prusty, A. B.·Truong, T. Q.·Dang, N. D. P.·Harder, S.·Lederbauer, J.·Lessel, I.·Ripin, N.·Kiel, C.·Fischer, U.·Kreienkamp, H.-J.·Lessel, D.

View original source ↗Export metadata ↓

Abstract

R-loops are three-stranded nucleic acid structures formed primarily during transcription that can obstruct replication fork progression, leading to replication stress and genome instability. Although RNA helicases are increasingly implicated in R-loop metabolism, how individual helicases safeguard genome integrity remains incompletely understood. Here we identify DHX30, a neurodevelopmental disorder-associated DExH-box RNA helicase, as a replication stress-responsive regulator of R-loops. We show that DHX30 localizes to the nucleus in addition to its known cytoplasmic and mitochondrial pools, and that its nuclear abundance in-creases following aphidicolin or camptothecin treatment. DHX30 deficiency results in RNA:DNA hybrid accumulation in human cells and zebrafish, R-loop-dependent DNA double-strand breaks, micronucleus formation, reduced DNA synthesis, and altered expression of immediate-early stress genes under basal conditions and following replication perturbation. Biochemically, DHX30 binds branched nucleic acid structures and directly unwinds R-loop substrates containing a 3' single-stranded RNA overhang in an ATP- and polarity-dependent manner. Proteomic analyses place DHX30 within a nuclear network of RNA- and R-loop-associated factors, including the RNA helicase DHX9. These findings establish DHX30 as a conserved R-loop-resolving helicase that protects from R-loop-induced replication perturbations, contributes to transcriptional homeostasis, and maintains genome stability, thereby providing a mechanistic framework for understanding DHX30-associated neurodevelopmental disorders.

Explore related subjects

molecular biology Preprint

Keep this discovery

Explore connections, maps & timelines

BibTeX RIS

Nedelcu, E. R., Yeo, N. C., Huber, H., Prusty, A. B., Truong, T. Q., Dang, N. D. P., Harder, S., Lederbauer, J., Lessel, I., Ripin, N., Kiel, C., Fischer, U., Kreienkamp, H.-J., Lessel, D.. 2026-10-27. DHX30 is a replication stress-responsive R-loop helicase that safeguards genome stability. https://doi.org/10.64898/2026.10.25.761626

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Endonuclease G governs an antiviral RNA surveillance system in budding yeast

Endonuclease G family DNA/RNA nucleases localize to the mitochondrial intermembrane space and prevent escape of mitochondrial DNA to the cytosol or nucleus. While extramitochondrial Endonuclease G also targets nuclear DNA during cell death, RNA regulatory roles of this family remain enigmatic. Here we discover an RNA surveillance system in Saccharomyces cerevisiae governed by Nuc1, the yeast Endonuclease G homolog. This system limits replication of L-A, an RNA mycovirus that endemically infects this organism. Whereas L-A resides in the cytosol, we find that Nuc1's antiviral function requires its mitochondrial intermembrane space localization. To understand Nuc1's indirect antiviral mechanism we investigated its genetic interactions with REX1 and REX2, RNA exonucleases that control 3' end processing of tRNA and rRNA intermediates in the nucleus. We demonstrate that the nuc1{triangleup} antiviral defect is massively amplified by rex1{triangleup} rex2{triangleup} leading to L-A RNA levels that surpass mature rRNAs and overwhelm the cells. NUC1 concurrently controls a pool of mis-processed tRNA and rRNA species that accumulate in rex1{triangleup} rex2{triangleup} mutants. Linking these mis-processed RNAs with L-A control, we show they are significantly reduced in L-A infected cells, suggesting that L-A depletes them to enhance its replication. Our findings identify a previously unrecognized RNA surveillance function of Nuc1 that prevents aberrant rRNA and tRNA species from promoting replication of the L-A mycovirus.

molecular biology↗

bioRxiv2026

Feedback control via H3K9 methylation and siRNA biogenesis autonomously maintains RNAi heterochromatin through continuous low-level transcription

The logic of RNA-based silencing involves nascent RNA molecules that serve as scaffolds to recruit proteins that repress transcription. Despite transcription and silencing being antagonistic, RNA-based recruitment mechanisms have been widely adopted in organisms ranging from yeast to humans. In fission yeast (Schizosaccharomyces pombe), the nuclear RNAi pathway that establishes heterochromatin at pericentromeric repeats to silence transcription exemplifies this paradox. Current models propose that cells reconcile these opposing activities by confining pericentromeric transcription to S phase while maintaining silencing during the rest of the cell cycle. Here, using strand-specific single-molecule RNA FISH (smFISH), we detect forward and reverse pericentromeric transcripts with high sensitivity and single-cell resolution. We find that repeat-associated transcription is rare, heterogeneous and happens independently of cell-cycle control. Instead, transcript production is governed solely by feedback involving small RNA biogenesis and H3K9 methylation. By combining mathematical modeling with experiments, we show that low levels of H3K9me or siRNAs prevent heterochromatin establishment, whereas high levels of H3K9me cause stochastic switching. Our work reveals that the same RNA-based feedback architecture can sample distinct transcriptional regimes, with feedback strength determining how often cells produce transcripts without any external regulatory inputs.

molecular biology↗

bioRxiv2026

Single-nucleus transcriptomics links telencephalic cell signatures to ocean acidification effects on social behaviour in a coral reef fish

Mutualistic interactions are fundamental to the health and stability of reef ecosystems. While elevated CO2 has been implicated in impairments in interaction behaviours, how this environmental change alters cellular states and intercellular communication in the brain remains unresolved. Here, by analysing the single-nucleus RNA profile of ~66,000 telencephalic nuclei from a keystone mutualist, the cleaner wrasse (Labroides dimidiatus), interacting under ambient or elevated CO2 conditions, we identify highly cell type specific neural responses to elevated CO2. We highlight a substantial depletion in Oligodendrocyte Precursor Cells (OPCs), notably linked to oligodendrocyte myelination and neural structure maintenance. Elevated CO2 also elicited targeted transcriptional shifts across discrete cell types, characterised by widespread glutamatergic signalling and altered co-expression networks associated with pH regulation, energetic metabolism, and circadian rhythm, thereby compromising the cleaner's physiologic equilibrium. Additionally, cell-cell communication was altered under elevated CO2, including remodelling in SEMA4 and PTPR signalling pathways within radial glial and OPCs, respectively, revealing network signalling rewiring in response to the changed environment. These findings uncover the high-resolution neural substrates vulnerable to ocean acidification, providing mechanistic insight into how climate-driven environmental changes alter brain function and destabilise complex mutualistic ecological interactions.

molecular biology↗