Credit:RNA damage compartmentalization by DHX9 stress granules.

RNA damage poses a significant threat to cellular function, necessitating robust mechanisms for repair and containment. While DNA damage has been extensively studied, the consequences of RNA damage in mammalian cells remain poorly understood. This study, led by Dr. Asifa Akhtar, sheds light on a novel cellular response to RNA damage by identifying a distinct type of stress granule (SG), marked by the dsRNA helicase DHX9, which plays a pivotal role in protecting daughter cells from parental RNA damage.

The study began by observing the intriguing behavior of the dsRNA helicase DHX9 during UVB and UVC irradiation. While DHX9 primarily resides in the nucleus under normal conditions, it was found to accumulate in cytoplasmic SGs upon exposure to these UV wavelengths. This observation was in contrast to other stress stimuli, suggesting a unique role for DHX9 in the context of UV-induced RNA damage.

To further investigate the nature of this phenomenon, the researchers employed a clever approach using 4-thiouridine (4sU), a uridine analog that can be incorporated into RNA but not DNA. By labeling cells with 4sU and subsequent exposure to UVA, they successfully induced RNA-protein and RNA-RNA crosslinking damage, recapitulating the accumulation of DHX9 SGs observed with UVB and UVC. This confirmed that RNA damage, specifically RNA crosslinking damage, is the molecular trigger for the formation of DHX9 SGs.

To further understand the composition and function of DHX9 SGs, the researchers developed a novel isolation method called FANCI (fluorescence-activated non-membrane condensates isolation). This method allowed them to purify intact SGs, including DHX9 SGs, with high purity and fidelity. RNA sequencing analysis of these purified SGs revealed a striking difference compared to classical SGs: while classical SGs are primarily composed of mature mRNA, DHX9 SGs are enriched in damaged intronic RNA. This finding suggested a novel role for DHX9 SGs in sequestering and processing damaged RNA.

Further investigation revealed that UV-induced intron damage disrupts proper RNA processing, leading to increased levels of abnormal splicing patterns and prolonged persistence of introns and pre-mRNAs. This disruption was observed in both classical and DHX9 SGs, highlighting a shared role for SGs in RNA processing during stress.

However, the study uncovered a crucial distinction between classical and DHX9 SGs in their response to intron damage. While classical SGs are formed downstream of translation arrest, DHX9 SGs actually promote cell survival and activate a dsRNA-related immune response and translation shutdown in daughter cells. This unique response suggests that DHX9 SGs play a protective role by mitigating the harmful effects of RNA damage and priming the immune response against potential viral infections.

The study further explored the role of DHX9 within the DHX9 SGs. By generating a cell line with a rapidly degradable DHX9 protein, the researchers demonstrated that DHX9 is critical for modulating dsRNA levels within the SGs. Depletion of DHX9 resulted in increased dsRNA levels and a heightened immune response, indicating that DHX9 helps maintain a balance between dsRNA levels and immune activation. In vitro phase separation assays confirmed that DHX9 preferentially forms condensates with dsRNA, suggesting a direct role in dsRNA processing and sequestration within the SGs.

The study also investigated the role of G3BP1/2, the canonical SG assembly factors, in DHX9 SG formation. By generating G3BP1/2 knockout cells, the researchers demonstrated that G3BP1/2 is essential for the assembly of DHX9 SGs. Depletion of G3BP1/2 prevented the formation of DHX9 SGs and blocked the dsRNA-related immune response and translation shutdown. This finding suggested that G3BP1/2 plays a unique role in the assembly and function of DHX9 SGs, distinct from their role in classical SGs.

Finally, the study explored the mechanisms of DHX9 SG disassembly. By analyzing the proteome of DHX9 SGs, the researchers identified the autophagy receptor p62 as a key component enriched within the SGs. Knockdown of p62 prevented the disassembly of DHX9 SGs, suggesting that autophagy plays a crucial role in clearing these granules. This finding adds another layer to the complexity of cellular stress responses, highlighting the interplay between stress granule formation and autophagy.

This study presents a groundbreaking discovery that expands our understanding of cellular stress responses and RNA damage mitigation. The identification of DHX9 SGs as a dedicated compartment for damaged intronic RNA and their unique role in activating a dsRNA-related immune response and translation shutdown opens up new avenues for research. Further investigation is needed to explore the molecular mechanisms by which DHX9 SGs are assembled, disassembled, and integrated into the broader cellular stress response.

One exciting direction is to investigate the potential for viral exploitation of DHX9 SGs. Some viruses have been shown to recruit DHX9 to cytoplasmic replication factories, potentially using this mechanism to evade the immune response. Understanding the molecular details of this process could provide valuable insights into viral pathogenesis and potential targets for antiviral therapies.

Additionally, exploring the role of DHX9 SGs in other contexts, such as DNA damage or cellular differentiation, could provide further insights into the broader significance of this novel stress granule type.

Overall, this study represents a significant advancement in our understanding of cellular stress responses and RNA damage mitigation. The discovery of DHX9 SGs opens up new avenues for research and provides a valuable framework for further investigation into the complex interplay between RNA damage, stress granules, and immune responses.

Reference:

1. Zhou Y, Panhale A, Shvedunova M, Balan M, Gomez-Auli A, Holz H, Seyfferth J, Helmstädter M, Kayser S, Zhao Y, Erdogdu NU, Grzadzielewska I, Mittler G, Manke T, Akhtar A. RNA damage compartmentalization by DHX9 stress granules. Cell. 2024 Mar 28;187(7):1701-1718.e28.