GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Series GSE198386 Query DataSets for GSE198386
Status Public on Oct 03, 2023
Title ADAR1 utilizes independent mechanisms to suppress MDA5 and PKR activation by cellular double-stranded RNA
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Summary The antiviral defense in vertebrates requires the innate immune system to sense foreign “non-self” nucleic acids while avoiding “self” nucleic acids, which is accomplished by an intricate system. Cellular double-stranded RNAs (dsRNAs) are edited by the RNA editing enzyme ADAR1 to prevent their dsRNA structure pattern from being recognized as viral dsRNA. Lack of RNA editing by ADAR1 enables activation of MDA5, a cytosolic dsRNA sensor, by cellular dsRNA. Additional RNA editing- independent functions of ADAR1 have been proposed, but the specific mechanism remains elusive. Here we demonstrate that RNA binding by ADAR1, independent of its editing activity, restricts the activation of PKR, another cytosolic dsRNA sensor, by cellular dsRNA. Mechanistically, the loss of ADAR1 editing caused MDA5 activation to induce interferon signaling, while a lack of ADAR1 protein or its dsRNA binding ability led to PKR activation, with subsequent stress granule formation and proliferation arrest. Based on these findings we rescued the Adar1−/− mice from embryonic lethality to adulthood by deleting both MDA5 and PKR, in contrast to the limited rescue of Adar1−/− mice by removing MDA5 or PKR alone. Our findings reveal a multifaceted contribution of ADAR1 in regulating the immunogenicity of “self” dsRNAs. Furthermore, ADAR1 is an immuno-oncology target for drug development, and the separation of ADAR1’s RNA editing and binding functions provides mechanistic insights for such developments. 
Overall design In order to study the effect of ADAR1 deficiency in human cells, HEK293T or U937 cells were transfected or infected with Cas9 and sgRNAs to make ADAR1 knockout (ADAR1KO) or ADAR1 editing deficient (ADAR1E912A) single clones. In HEK293T, wild type (WT), ADAR1KO, and ADAR1E912A cells were harvest and subjected to stranded, paired-end RNAseq. In U937, WT or ADAR1KO cells were treated with or without 50nM phorbol myristate acetate for three days to induce the differentiation of U937 cells to macrophages. After that, the cells were subjected to stranded, paired-end RNAseq. In order to identify the factors accounting for IFN-induced cell growth arrest in the absence of ADAR1, U937 ADAR1KO cells were infected with human CRISPR gRNA library followed by IFN treatment for 14 days. The genomic DNA was then purified for gRNA sequencing.  der to identify PKR-enriched endogenous RNAs, flag-PKR was overexpressed in WT and ADAR1 KO HEK293T cells . Then, flag-PKR native RNA IP was performed to enrich RNAs. Flag-PKR-enriched RNAs were sequenced.
Contributor(s) Shibin H
Citation missing Has this study been published? Please login to update or notify GEO.
Submission date Mar 10, 2022
Last update date Oct 03, 2023
Contact name Shibin Hu
Organization name Stanford University
Department Genetics
Lab Jin Billy Li
Street address 240 pasteur drive, 4300
City Stanford
State/province CA
ZIP/Postal code 94305
Country USA
Platforms (2)
GPL20301 Illumina HiSeq 4000 (Homo sapiens)
GPL21697 NextSeq 550 (Homo sapiens)
Samples (17)
GSM5946006 HEK293T_WT_1
GSM5946007 HEK293T_WT_2
GSM5946008 HEK293T_ADAR1E912A_1
BioProject PRJNA814746

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE198386_ADAR1_rawcount.txt.gz 512.7 Kb (ftp)(http) TXT
GSE198386_CRISPRscreen_rawcount.txt.gz 2.3 Mb (ftp)(http) TXT
GSE198386_Flag_PKR_RIP_TPM.csv.gz 143.4 Kb (ftp)(http) CSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap