Adenosine deaminases acting on RNA (ADARs) are editing enzymes that convert adenosine to inosine in duplex RNA, a modification reaction with wide-ranging consequences in RNA function. Understanding of the ADAR reaction mechanism, the origin of editing-site selectivity, and the effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. Here we describe four crystal structures of the human ADAR2 deaminase domain bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis of the ADAR deaminase domain's dsRNA specificity, its base-flipping mechanism, and its nearest-neighbor preferences. In addition, we identified an ADAR2-specific RNA-binding loop near the enzyme active site, thus rationalizing differences in selectivity observed between different ADARs. Finally, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease.

Adenosine deaminases acting on RNA (ADARs) are editing enzymes that convert adenosine to inosine in duplex RNA, a modification reaction with wide-ranging consequences in RNA function. Understanding of the ADAR reaction mechanism, the origin of editing-site selectivity, and the effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. Here we describe four crystal structures of the human ADAR2 deaminase domain bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis of the ADAR deaminase domain's dsRNA specificity, its base-flipping mechanism, and its nearest-neighbor preferences. In addition, we identified an ADAR2-specific RNA-binding loop near the enzyme active site, thus rationalizing differences in selectivity observed between different ADARs. Finally, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease.

KRAS is a regulator of the nutrient stress response in non-small-cell lung cancer (NSCLC). Induction of the ATF4 pathway during nutrient depletion requires AKT and NRF2 downstream of KRAS. The tumor suppressor KEAP1 strongly influences the outcome of activation of this pathway during nutrient stress; loss of KEAP1 in KRAS mutant cells leads to apoptosis. Through ATF4 regulation, KRAS alters amino acid uptake and asparagine biosynthesis. The ATF4 target asparagine synthetase (ASNS) contributes to apoptotic suppression, protein biosynthesis, and mTORC1 activation. Inhibition of AKT suppressed ASNS expression and, combined with depletion of extracellular asparagine, decreased tumor growth. Therefore, KRAS is important for the cellular response to nutrient stress, and ASNS represents a promising therapeutic target in KRAS mutant NSCLC.