RESUMO
RIG-I is a DExD/H-box RNA helicase and functions as a critical cytoplasmic sensor for RNA viruses to initiate antiviral interferon (IFN) responses. Here we demonstrate that another DExD/H-box RNA helicase DHX36 is a key molecule for RIG-I signaling by regulating double-stranded RNA (dsRNA)-dependent protein kinase (PKR) activation, which has been shown to be essential for the formation of antiviral stress granule (avSG). We found that DHX36 and PKR form a complex in a dsRNA-dependent manner. By forming this complex, DHX36 facilitates dsRNA binding and phosphorylation of PKR through its ATPase/helicase activity. Using DHX36 KO-inducible MEF cells, we demonstrated that DHX36 deficient cells showed defect in IFN production and higher susceptibility in RNA virus infection, indicating the physiological importance of this complex in host defense. In summary, we identify a novel function of DHX36 as a critical regulator of PKR-dependent avSG to facilitate viral RNA recognition by RIG-I-like receptor (RLR).
Assuntos
RNA Helicases DEAD-box/imunologia , Infecções por Vírus de RNA/imunologia , Transdução de Sinais/imunologia , eIF-2 Quinase/imunologia , Grânulos Citoplasmáticos/imunologia , Proteína DEAD-box 58 , RNA Helicases DEAD-box/metabolismo , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Técnicas de Inativação de Genes , Células HEK293 , Células HeLa , Humanos , Imunoprecipitação , Vírus de RNA/imunologia , RNA de Cadeia Dupla/imunologia , RNA Interferente Pequeno/genética , RNA Viral/imunologia , Receptores Imunológicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse Fisiológico , TransfecçãoRESUMO
The DEAH helicase RHAU (alias DHX36, G4R1) is the only helicase shown to have G-quadruplex (G4)-RNA resolvase activity and the major source of G4-DNA resolvase activity. Previous report showed RHAU mRNA expression to be elevated in human lymphoid and CD34(+) BM cells, suggesting a potential role in hematopoiesis. Here, we generated a conditional knockout of the RHAU gene in mice. Germ line deletion of RHAU led to embryonic lethality. We then targeted the RHAU gene specifically in the hematopoiesis system, using a Cre-inducible system in which an optimized variant of Cre recombinase was expressed under the control of the Vav1 promoter. RHAU deletion in hematopoietic system caused hemolytic anemia and differentiation defect at the proerythroblast stage. The partial differentiation block of proerythroblasts was because of a proliferation defect. Transcriptome analysis of RHAU knockout proerythroblasts showed that a statistically significant portion of the deregulated genes contain G4 motifs in their promoters. This suggests that RHAU may play a role in the regulation of gene expression that relies on its G4 resolvase activity.