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posted on 2024-04-04, 14:20 authored by Kyle A. Cottrell, Sua Ryu, Jackson R. Pierce, Luisangely Soto Torres, Holly E. Bohlin, Angela M. Schab, Jason D. Weber Rescue of PKR activation by DHX9 mutants in SK-BR-3. A, Schematic showing the domain structure of ADAR1 isoforms, DHX9 and mutants of DHX9, dsRBD refers to the dsRNA binding domain. B, Representative immunoblot showing the phenotype of ADAR1 and DHX9 knockdown with DHX9, DHX9K417R or dsRBD-EGFP overexpression in SK-BR-3. Immunoblots for other replicates and uncropped blots can be found in Source Data Figures. C, Fold change of PKR phosphorylation at Thr-446 upon ADAR1 and DHX9 knockdown with DHX9, DHX9K417R or dsRBD-EGFP overexpression in SK-BR-3, quantified from immunoblots in B. Quantification of protein expression for other proteins of interest can be found in Supplementary Fig. S8B–S8F. D, Representative foci formation phenotype of ADAR1 and DHX9 knockdown with DHX9, DHX9K417R or dsRBD-EGFP overexpression in SK-BR-3. Quantification of relative foci area is shown in E. Timepoints for collecting protein lysates and foci formation are the same as described in Fig. 4 for SK-BR-3. Bars represent the average of at least three replicates, error bars are ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001. P values determined by Dunnett test.
Funding
HHS | National Institutes of Health (NIH)
U.S. Department of Defense (DOD)
History
ARTICLE ABSTRACT
Detection of viral double-stranded RNA (dsRNA) is an important component of innate immunity. However, many endogenous RNAs containing double-stranded regions can be misrecognized and activate innate immunity. The IFN-inducible ADAR1-p150 suppresses dsRNA sensing, an essential function for adenosine deaminase acting on RNA 1 (ADAR1) in many cancers, including breast. Although ADAR1-p150 has been well established in this role, the functions of the constitutively expressed ADAR1-p110 isoform are less understood. We used proximity labeling to identify putative ADAR1-p110–interacting proteins in breast cancer cell lines. Of the proteins identified, the RNA helicase DHX9 was of particular interest. Knockdown of DHX9 in ADAR1-dependent cell lines caused cell death and activation of the dsRNA sensor PKR. In ADAR1-independent cell lines, combined knockdown of DHX9 and ADAR1, but neither alone, caused activation of multiple dsRNA sensing pathways leading to a viral mimicry phenotype. Together, these results reveal an important role for DHX9 in suppressing dsRNA sensing by multiple pathways.
These findings implicate DHX9 as a suppressor of dsRNA sensing. In some cell lines, loss of DHX9 alone is sufficient to cause activation of dsRNA sensing pathways, while in other cell lines DHX9 functions redundantly with ADAR1 to suppress pathway activation.
