Alter ego: RNA helicase is recruited to sites of DNA damage

From the Warren Lab, Clinical Research Division

The RNA helicase DDX3X spends its days unwinding RNA:RNA duplexes and binding key mediators of RNA metabolism. A regulator of RNA transcription, splicing, nuclear export, and translation, DDX3X plays an essential role in the processing and delivery of a ceaseless flow of messages between the blueprints and the builders of the cell. A noble, if grinding, occupation. If you pay close attention, however, you’ll find that there is more to this helicase than initially meets the eye. When trouble arises in the cell, such as cellular stress, inflammation, and metabolic strain, DDX3X leaps into action. A key decider in matters of life and death for the cell, this helicase regulates processes as diverse as stress granule assembly (*BAM*), inflammasome activation (*ZAP*), cytokine production (*BOOM*), the initiation of antiviral immune responses (*POW*), and apoptosis (*BANG*). If that isn’t impressive enough, DDX3X also plays roles in embryogenesis, including regulation of WNT/β-catenin signaling. Addressing mortal threats and fostering proper youth development, DDX3X is a true molecular hero. However, with great power comes great responsibility. Aberrant disruption or alteration of DDX3X function is associated with a litany of disease states, including developmental, neurological, and malignant conditions, and many viruses are known to exploit DDX3X to promote their own replication.

Intriguing evidence for yet another role of DDX3X has emerged, this time in the context of the DNA damage response. Disruption of this helicase impairs DNA repair pathways, leading to an accumulation of DNA breaks. The involvement of RNA and RNA-binding proteins in the DNA damage response is an area of growing scientific interest. DDX3X has known roles in controlling the expression of genes involved in DNA repair, but could it take part more directly in the DNA damage response? Members of the Warren Laboratory in the Fred Hutch Clinical Research Division, led by postdoctoral fellow Dr. Michael Cargill, investigated the role of DDX3X in the DNA damage response. Their work, recently published in DNA Repair, reveals that DDX3X is actively recruited to sites of DNA damage, providing evidence for direct involvement in DNA repair beyond transcriptional regulation of repair factors.

RNA metabolism regulator DDX3X accumulates at sites of DNA damage in live cells, dependent on PARP1 activity.
RNA metabolism regulator DDX3X accumulates at sites of DNA damage in live cells, dependent on PARP1 activity. Image provided by Dr. Cargill.

To begin to examine the activities of DDX3X in the nucleus, the Warren lab first performed protein immunoprecipitation to identify associated protein partners. Encouragingly, they found that DDX3X precipitated with factors involved in RNA metabolism and splicing factors, as well as with DNA repair proteins. Next, the researchers induced DNA damage in cells by administering gamma irradiation, and assessed the localization of DDX3X through three-dimensional imaging of fixed cells. They observed that the helicase localized in foci within the nucleus, colocalizing with established markers of DNA damage (γH2AX and 53BP1). Finally, the Warren group utilized an inducible restriction enzyme-based system to introduce targeted DNA double-strand breaks, followed by chromatin profiling (CUT&RUN) sequencing to detect enrichment of DDX3X at predicted cut sites. Indeed, DDX3X was enriched at DNA breaks following restriction enzyme cutting, further substantiating their hypothesis that this helicase might have direct involvement in the DNA damage response.

Having established DDX3X localization at DNA breaks in fixed cells, the group sought to track the recruitment of the helicase in live cells. To visualize DDX3X protein, they utilized a green fluorescent protein (GFP)-tag to track its location. To facilitate more robust detection of DDX3X by live imaging, the researchers mutated two residues in its nuclear export signal sequence (DDX3XL19A/L21A) resulting in increased nuclear concentrations. Next, they developed a system for inducing focal DNA damage by micro-irradiating live cells and visualizing DNA damage via fluorescently tagged Ku70, a DNA repair protein that binds to broken DNA strand ends. Here, the Warren group confirmed that both DDX3X-GFP and DDX3XL19A/L21A -GFP co-localized at DNA damage sites in this setting. Kinetic analysis revealed that co-localization of DDX3X with Ku70 at DNA breaks peaked at ~90 seconds post-irradiation.

Finally, the authors began to investigate the mechanisms of recruitment of DDX3X to sites of DNA damage. DDX3X is known to form stress granules through its intrinsically disordered domains (IDDs) in a process similar to the recruitment of RNA-binding proteins to damaged DNA. They produced IDD-deleted DDX3X mutants and, using their live cell imaging system, observed that deletion of its IDDs completely abrogated recruitment of the helicase to sites of DNA damage. Lastly, the Warren group probed the role of PARP1, a DNA repair protein that facilitates the precipitation of other RNA-binding proteins at sites of DNA damage, in the localization of DDX3X at DNA breaks. Pharmacological inhibition, genetic disruption, and catalytic mutation of PARP1 each abrogated DDX3X recruitment, supporting a mechanism by which the helicase is recruited to sites of DNA damage through its IDDs in a PARP1-dependent manner.

Together, these findings implicate a direct role for DDX3X in the DNA damage response – a more intimate involvement than the previously characterized transcriptional regulation of repair factors. “This protein unwinds tangled RNA and is canonically known as a player in gene expression. Our study highly suggests this protein is also involved with the repair of DNA damage,” explained Dr. Cargill. “This creates several exciting questions: How does DDX3X affect DNA repair? Is DDX3X specifically recruited to help repair regions of transcription?” Further, these studies could yield important findings for the field of oncology. “DDX3X is frequently mutated in diseases such as Burkitt lymphoma and medulloblastoma,” said Dr. Cargill. “Does this novel role relate to the development of disease when mutated?” Stay tuned for more adventures of DDX3X…

This work was funded by the National Institutes of Health and the Cancer Therapeutics Endowment.

UW/Fred Hutch Cancer Consortium member Edus Warren contributed to this work.

Cargill MJ, Morales A, Ravishankar S, Warren EH. RNA helicase, DDX3X, is actively recruited to sites of DNA damage in live cells. DNA Repair (Amst). 2021 Jul;103:103137. doi: 10.1016/j.dnarep.2021.103137. Epub 2021 May 18. PMID: 34083132.