The effect of ADAR1 depletion on mitochondrial proteins
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ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture
SUBMITTER: 
Hachung Chung
LAB HEAD: Hachung Chung
PROVIDER: PXD069896 | Pride | 2026-01-23
REPOSITORIES: Pride
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Project description:Endogenous (self) double-stranded RNAs (dsRNAs) in human cells can activate innate immune responses. ADAR1, an A-to-I editing enzyme of dsRNAs, suppresses aberrant immune activation by self-dsRNAs. However, how ADAR1 influences the cellular dsRNA landscape remains unclear. We show that human ADAR1 downregulates self-dsRNA abundance through editing-dependent and editing-independent mechanisms. We further conducted quantitative dsRNA-sequencing on wild-type and ADAR1-deficient cells. dsRNAs are enriched in protein-coding mRNAs − especially those with repetitive elements and elongated 3’UTRs − and mitochondrial RNAs. ADAR1 loss markedly increased dsRNA quantity but minimally impacted dsRNA identity. ADAR1-regulated dsRNA transcripts consist of nuclear-encoded mRNAs and, unexpectedly, mitochondria-encoded RNAs rarely edited by ADAR1. Accordingly, dsRNAs accumulate to high levels within the mitochondria of ADAR1-deficient cells. Notably, ADAR1 loss sensitizes cells to inflammation under mitochondrial stress (e.g., mitochondrial herniation, X-ray radiation). Hence, we show that dsRNAs regulated by ADAR1 go beyond A-to-I edited transcripts, and that ADAR1 can control mitochondrial-dsRNAs.
2026-01-13 | GSE293278 | GEO
Project description:The RNA editing enzyme ADAR1, is essential for correct functioning of innate immune responses. The ADAR1p110 isoform is mainly nuclear and ADAR1p150, which is interferon inducible, is predominately cytoplasmic. Using three different approaches, co-IP with endogenous ADAR1, and Strep-tag co-IP and BioID with individual ADAR1 isoforms, a comprehensive interactome was generated during both homeostasis and the IFN response. Both known and novel interactors and editing regulators were identified. Nuclear proteins were detected as stable interactors with both ADAR1 isoforms. In contrast, BioID identified distinct protein networks for each ADAR1 isoform, with nuclear components observed with ADAR1p110 and components of cytoplasmic cellular condensates with ADAR1p150. RNase A digestion distinguished between distal and proximal interactors, as did a dsRNA-binding mutant of ADAR1 which demonstrated the importance of dsRNA binding for ADAR1 interactions. IFN treatment did not affect the core ADAR1 interactomes but resulted in novel interactions, the majority of which are proximal interactions retained after RNase A treatment. Short treatment with HMW poly(I:C) during the IFN response resulted in dsRNA-binding-dependent changes in the proximal protein network of ADAR1p110 and association of the ADAR1p150 proximal protein network with some components of antiviral stress granules.
2024-12-08 | PXD056080 | Pride
Project description:Small non-coding RNA biogenesis typically involves cleavage of structured precursors by RNase III-like endonucleases. However, guide RNAs that direct U-insertion/deletion mRNA editing in mitochondria of trypanosomes maintain 5â² triphosphate characteristic of transcription start site and possess U-tail indicative of 3â² processing and uridylation. Here, we identified a protein complex composed of RET1 TUTase and 3â²-5â² DSS1 exonuclease, and three additional subunits. This complex, termed mitochondrial 3â² processome (MPsome), is responsible for primary uridylation of ~800-nt gRNA precursors, their processive degradation to a mature length of 50-60 nt, and secondary U-tail addition. Both strands of gRNA gene are transcribed giving rise to sense and antisense precursors of similar size. Head-to-head hybridization of these transcripts blocks symmetrical 3â²-5â² degradation at the fixed distance from the double-stranded region. Together, our findings suggest a model in which gRNA is derived from the 5â² extremity of a primary molecule by uridylation-induced, antisense transcription-controlled exonucleolytic degradation. 1. we first sequenced guide RNA precursor (Gel-fractioned total cellular RNA 600-1500nt was used) transcripts from three replicates to study the their tail features and also validate observation of sense/antisense accumulation upon perturbation of pre-processing complex based on few cases. 2. we then sequenced mitochondrial small RNA and built a reference for small RNAs using our custom algorithm. We then took the mitochondrial small RNA data and uncovered the sense/antisense pair. 3. We then used CLIP-Seq data to investigate in vivo binding sites and, together with RNA IP-Seq data to understand what determine the relative abundance of sense and antisense pair of the duplex. 4. We used sequenced small mitochondrial RNA in different RNAi experiments (For RNAi experiments, 30-70nt RNA fraction was gel-isolated from total cellular RNAs) to understand which protein will affect the Utail length in mature small mitochondrial RNA.
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Project description: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.
2023-10-03 | GSE198386 | GEO
Project description:Endogenous double-stranded RNA (dsRNA) triggers pro-inflammatory signaling and has been shown to accumulate in Alzheimer’s disease (AD), but the origins of this dsRNA are incompletely understood. Transposable elements (TEs), which are non-coding DNA sequences capable of forming dsRNA, are a potential endogenous source of dsRNA in AD. We generated dsRNA immunoprecipitation (J2 dsRNA antibody) data on astrocytes treated with or without a siRNA to knock down the dsRNA editing enzyme ADAR1. We analyzed TE transcripts enriched in dsRNA pools when ADAR1 was knocked down, and we identified putative dsRNA-prone TEs, which were also present in secondary analyses of datasets profiling RNAs bound to PKR, MDA5, and the p110 isoform of ADAR1.
2025-07-25 | GSE303455 | GEO
Project description:Adenosine deaminase acting on RNA 1 (ADAR1), is an enzyme that catalyzes the conversion of adenosine to inosine in double-stranded RNA, a process critical for regulation of innate immune response and distinguishing between ‘self and non-self RNA’. It is expressed as two isoforms: nucleolar p110 and cytoplasmic, interferon (IFN)-inducible, p150. The interactome of the p110 under steady-state conditions is well-studied; however, less is known about the interactions of the p150 isoform, particularly during IFN response. To elucidate ADAR1's protein interactions during IFN stimulation, alongside steady-state conditions, three distinct methods of enrichment were used followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These included: immunoprecipitation (IP) of endogenous ADAR1, IP of Strep II-tagged ADAR1, and proximity labeling using BioID. Individual ADAR1 isoforms (p110 and p150) and their respective dsRNA binding-deficient mutants were created to discern isoform-specific and dsRNA-dependent interactions. Altogether, our results reveal a comprehensive ADAR1 interaction map, identifying both known and novel partners, and highlighting the isoform-specific and dsRNA-binding-dependent nature of ADAR1 interactions. Under IFN stimulation, ADAR1's interaction spectrum encompasses viral replication inhibitors and LINE-1 regulators. Mimicking viral infection with HMW poly(I:C) changed the proximal network of proteins for both isoforms. Our findings provide new insights into ADAR1's roles and its dynamic during IFN response.
2024-12-05 | PXD049681 | Pride
Project description:Adenosine deaminase acting on RNA 1 (ADAR1), is an enzyme that catalyzes the conversion of adenosine to inosine in double-stranded RNA, a process critical for regulation of innate immune response and distinguishing between ‘self and non-self RNA’. It is expressed as two isoforms: nucleolar p110 and cytoplasmic, interferon (IFN)-inducible, p150. The interactome of the p110 under steady-state conditions is well-studied; however, less is known about the interactions of the p150 isoform, particularly during IFN response. To elucidate ADAR1's protein interactions during IFN stimulation, alongside steady-state conditions, three distinct methods of enrichment were used followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These included: immunoprecipitation (IP) of endogenous ADAR1, IP of Strep II-tagged ADAR1, and proximity labeling using BioID. Individual ADAR1 isoforms (p110 and p150) and their respective dsRNA binding-deficient mutants were created to discern isoform-specific and dsRNA-dependent interactions. Altogether, our results reveal a comprehensive ADAR1 interaction map, identifying both known and novel partners, and highlighting the isoform-specific and dsRNA-binding-dependent nature of ADAR1 interactions. Under IFN stimulation, ADAR1's interaction spectrum encompasses viral replication inhibitors and LINE-1 regulators. Mimicking viral infection with HMW poly(I:C) changed the proximal network of proteins for both isoforms. Our findings provide new insights into ADAR1's roles and its dynamic during IFN response.
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Project description:Adenosine deaminase acting on RNA 1 (ADAR1), is an enzyme that catalyzes the conversion of adenosine to inosine in double-stranded RNA, a process critical for regulation of innate immune response and distinguishing between ‘self and non-self RNA’. It is expressed as two isoforms: nucleolar p110 and cytoplasmic, interferon (IFN)-inducible, p150. The interactome of the p110 under steady-state conditions is well-studied; however, less is known about the interactions of the p150 isoform, particularly during IFN response. To elucidate ADAR1's protein interactions during IFN stimulation, alongside steady-state conditions, three distinct methods of enrichment were used followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These included: immunoprecipitation (IP) of endogenous ADAR1, IP of Strep II-tagged ADAR1, and proximity labeling using BioID. Individual ADAR1 isoforms (p110 and p150) and their respective dsRNA binding-deficient mutants were created to discern isoform-specific and dsRNA-dependent interactions. Altogether, our results reveal a comprehensive ADAR1 interaction map, identifying both known and novel partners, and highlighting the isoform-specific and dsRNA-binding-dependent nature of ADAR1 interactions. Under IFN stimulation, ADAR1's interaction spectrum encompasses viral replication inhibitors and LINE-1 regulators. Mimicking viral infection with HMW poly(I:C) changed the proximal network of proteins for both isoforms. Our findings provide new insights into ADAR1's roles and its dynamic during IFN response.
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Project description:Adenosine deaminase acting on RNA 1 (ADAR1), is an enzyme that catalyzes the conversion of adenosine to inosine in double-stranded RNA, a process critical for regulation of innate immune response and distinguishing between ‘self and non-self RNA’. It is expressed as two isoforms: nucleolar p110 and cytoplasmic, interferon (IFN)-inducible, p150. The interactome of the p110 under steady-state conditions is well-studied; however, less is known about the interactions of the p150 isoform, particularly during IFN response. To elucidate ADAR1's protein interactions during IFN stimulation, alongside steady-state conditions, three distinct methods of enrichment were used followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). These included: immunoprecipitation (IP) of endogenous ADAR1, IP of Strep II-tagged ADAR1, and proximity labeling using BioID. Individual ADAR1 isoforms (p110 and p150) and their respective dsRNA binding-deficient mutants were created to discern isoform-specific and dsRNA-dependent interactions. Altogether, our results reveal a comprehensive ADAR1 interaction map, identifying both known and novel partners, and highlighting the isoform-specific and dsRNA-binding-dependent nature of ADAR1 interactions. Under IFN stimulation, ADAR1's interaction spectrum encompasses viral replication inhibitors and LINE-1 regulators. Mimicking viral infection with HMW poly(I:C) changed the proximal network of proteins for both isoforms. Our findings provide new insights into ADAR1's roles and its dynamic during IFN response.
2024-12-05 | PXD049689 | Pride
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