Abstract
BackgroundNuclear factor-κB (NF-κB) plays a prominent role in promoting inflammation and resistance to DNA damaging therapy. We searched for proteins that modulate the NF-κB response as a prerequisite to identifying novel factors that affect sensitivity to DNA damaging chemotherapy.ResultsUsing streptavidin-agarose pull-down, we identified the DExD/H-box RNA helicase, DDX39B, as a factor that differentially interacts with κB DNA probes. Subsequently, using both RNA interference and CRISPR/Cas9 technology, we demonstrated that DDX39B inhibits NF-κB activity by a general mechanism involving inhibition of p65 phosphorylation. Mechanistically, DDX39B mediates this effect by interacting with the pattern recognition receptor (PRR), LGP2, a pathway that required the cellular response to cytoplasmic double-stranded RNA (dsRNA). From a functional standpoint, loss of DDX39B promoted resistance to alkylating chemotherapy in glioblastoma cells. Further examination of DDX39B demonstrated that its protein abundance was regulated by site-specific sumoylation that promoted its poly-ubiquitination and degradation. These post-translational modifications required the presence of the SUMO E3 ligase, PIASx-β. Finally, genome-wide analysis demonstrated that despite the link to the PRR system, DDX39B did not generally inhibit interferon-stimulated gene expression, but rather acted to attenuate expression of factors associated with the extracellular matrix, cellular migration, and angiogenesis.ConclusionsThese results identify DDX39B, a factor with known functions in mRNA splicing and nuclear export, as an RNA-binding protein that blocks a subset of the inflammatory response. While these findings identify a pathway by which DDX39B promotes sensitization to DNA damaging therapy, the data also reveal a mechanism by which this helicase may act to mitigate autoimmune disease.
Highlights
Nuclear factor-κB (NF-κB) plays a prominent role in promoting inflammation and resistance to DNA damaging therapy
The lack of increased double-stranded RNA (dsRNA) suggested that simple protection of cytoplasmic mRNA is not the mechanism by which DDX39B attenuates NF-κB, a finding supported by the observation that neither Rig-I nor Mda5 were involved in this response
DDX39B is best known for its role in regulating mRNA splicing and nuclear export, independent studies identified this helicase as Bat1, a factor associated with inflammatory diseases [18,19,20,21]
Summary
Nuclear factor-κB (NF-κB) plays a prominent role in promoting inflammation and resistance to DNA damaging therapy. Among the many inducing stimuli, factors such as cytosolic nucleic acid activate NF-κB as part of the innate immune system [3] While this response is essential for combating exogenous pathogens, endogenously produced nucleic acids can activate NF-κB signaling. Such endogenous activation can lead to chronic inflammation that promotes autoimmune disease, cancer, and resistance to therapy [4, 5]. Given the harmful effects of un-controlled NF-κB activity and inflammation, a variety of mechanisms have evolved to prevent excess activation of NF-κB by endogenous sources [6] In this regard, recent studies have highlighted the role of RNA-binding proteins (RBPs) in preventing inflammation due to endogenously produced RNA [7, 8]
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