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RNA editing is the essential function of ADAR1 and, in the absence of MDA5, is dispensable for normal adult homeostasis [RNAseq (Adult Brain)]

ABSTRACT: Adenosine-to-Inosine (A-to-I) editing of dsRNA by ADAR proteins is a pervasive feature of the epitranscriptome. There are estimated to be over 100 million potential A-to-I editing sites in humans and A-to-I editing can have varying consequences for gene expression. Whilst editing resulting in protein recoding defines the role of ADAR2, ADAR1 has been proposed to have both editing-dependent and -independent functions. The relative contribution of these putative functions to ADAR1 biology is unclear. We demonstrate that the absence of ADAR1-mediated editing is well tolerated when the cytosolic dsRNA sensor MDA5 is deleted. These mice have normal hematopoiesis, tissue patterning and life span. A direct comparison of the complete deletion of ADAR1 and the specific loss of A-to-I editing activity demonstrates that RNA editing is the only essential function of ADAR1 in adult mice. Therefore, preventing MDA5 substrate formation by endogenous RNA is the essential in vivo function of ADAR1-mediated editing.

ORGANISM(S): Mus musculus

PROVIDER: GSE94387 | GEO | 2017/08/14

SECONDARY ACCESSION(S): PRJNA369545

REPOSITORIES: GEO

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Project description:Sensing of double-stranded RNA (dsRNA) is an important component of innate immunity. Proteins like PKR and MDA5 recognize dsRNA and activate various pathways to fight viral infection. In addition to viral dsRNA, many endogenous RNAs containing double-stranded regions can be misrecognized as immunogenic. The RNA editing enzyme ADAR1, specifically its p150 isoform, has been shown to suppress activation of PKR and MDA5 through its A-to-I editing activity. While the cytoplasmic ADAR1-p150 isoform has been well established within this role, the functions of the nuclear ADAR1-p110 isoform are less understood. To address this knowledge gap, we utilized proximity labeling by APEX2 to identify putative ADAR1-p110 interacting proteins. We identified 110 proteins across three breast cancer cell lines that either interact with p110 or are within close proximity. Many of these proteins have known roles in RNA metabolism. Nine of the identified proteins belong to the DEAD or DEAH box families of RNA helicases, including DHX9. DHX9 is overexpressed in breast cancer, with expression closely correlated with that of p110. By co-immunoprecipitation we confirmed that p110 interacts with DHX9. Knockdown of DHX9 in several triple-negative breast cancer cell lines caused cell death and activation of the dsRNA sensor PKR. In two cell lines that are refractory to knockdown of ADAR1, the combined knockdown of DHX9 and ADAR1 caused a substantial increase in PKR activity, where knockdown of either alone had no effect. Knockdown of DHX9 and ADAR1 caused activation of the type I IFN pathway, RNase L and NF-KB signaling. Activation of these proteins and pathways was not seen with individual knockdown of ADAR1 or DHX9. Activation of PKR following combined knockdown of ADAR1 and DHX9 could be rescued by expression of p110, p150, DHX9, and catalytically inactive DHX9. Additionally PKR activation could be rescued by the dsRBM of DHX9, revealing an important role for dsRBMs in suppressing PKR activation. Together these results reveal an important role for DHX9 in suppressing dsRNA sensing by multiple pathways.

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RNA editing is the essential function of ADAR1 and, in the absence of MDA5, is dispensable for normal adult homeostasis [RNAseq (Adult Brain)]

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