The Ubiquitin Ligase SMURF1 Catalyzes the Polyubiquitination of ADAR1 / 中国生物化学与分子生物学报

It is known that SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) mediates autophagy through its E3 ubiquitin ligase activity, but the ubiquitinated substrates of SMURF1 need to be further explored. In this paper, the interacting proteins of SMURF1 in THP-1 cells were captured and identified by co-immunoprecipitation (Co-IP) combined with mass spectrometry. It was found that SMURF1 could physically bind to 222 proteins in THP-1 cells, and Adenosine deaminase acting on RNA 1 (ADAR1) had a higher peptide binding score. SMURF1 overexpression vectors were constructed and transfected into HEK-293T cells, then Co-IP and Western blotting assays verified the interaction between exogenous SMURF1 and endogenous ADAR1. qRT-PCR and Western blotting assays were carried out after transfecting SMURF1 overexpression vectors in HEK-293T cells, which identified that overexpression of SMURF1 attenuated the protein levels of ADAR1 (P<0. 05). However, there was no significant difference in the mRNA level of ADAR1. HEK-293T cells with normal and overexpressing SMURF1 were treated with cycloheximide (CHX), respectively, and Western blotting assays showed a shortened half-life of ADAR1 after overexpression of SMURF1 (P < 0. 05). Furthermore, overexpression of SMURF1 increased the polyubiquitination level of ADAR1 as detected by Co-IP and Western blot (P<0. 05). After the proteasome inhibitor (MG132) treatment, the Western blotting assay was performed to demonstrate that the negative regulatory effect of SMURF1 on ADAR1 was weakened after the proteasome degradation pathway was attenuated (P<0. 05). This study shows that SMURF1 interacts with ADAR1, catalyzes the polyubiquitination of ADAR1 and mediates its degradation through the proteasome pathway, which provides a theoretical basis for exploring the various biological functions of SMURF1 by affecting the stability of ADAR1.

ADAR1 regulates vascular remodeling in hypoxic pulmonary hypertension through N1-methyladenosine modification of circCDK17

Pulmonary hypertension (PH) is an extremely malignant pulmonary vascular disease of unknown etiology. ADAR1 is an RNA editing enzyme that converts adenosine in RNA to inosine, thereby affecting RNA expression. However, the role of ADAR1 in PH development remains unclear. In the present study, we investigated the biological role and molecular mechanism of ADAR1 in PH pulmonary vascular remodeling. Overexpression of ADAR1 aggravated PH progression and promoted the proliferation of pulmonary artery smooth muscle cells (PASMCs). Conversely, inhibition of ADAR1 produced opposite effects. High-throughput whole transcriptome sequencing showed that ADAR1 was an important regulator of circRNAs in PH. CircCDK17 level was significantly lowered in the serum of PH patients. The effects of ADAR1 on cell cycle progression and proliferation were mediated by circCDK17. ADAR1 affects the stability of circCDK17 by mediating A-to-I modification at the A5 and A293 sites of circCDK17 to prevent it from m1A modification. We demonstrate for the first time that ADAR1 contributes to the PH development, at least partially, through m1A modification of circCDK17 and the subsequent PASMCs proliferation. Our study provides a novel therapeutic strategy for treatment of PH and the evidence for circCDK17 as a potential novel marker for the diagnosis of this disease.

Four novel mutations of ADAR1 in Chinese patients with dyschromatosis symmetrica hereditaria

Background: Novel mutations in adenosine deaminase acting on RNA 1 gene (ADAR1) are responsible for dyschromatosis symmetrica hereditaria (DSH). DSH patients display a mixture of hyperpigmented and hypopigmented macules on the dorsal aspects of the extremities, and freckle-like macules on the face. Aims: To provide new evidence for further study of the etiopathogenisis of DSH. Methods: Genomic DNA was extracted and used as a template for the polymerase chain reaction (PCR) amplification of all 15 coding exons as well as intron-exon boundaries of ADAR1. The PCR products were sequenced directly. Results: We identified eight mutations of ADAR1 in four Chinese pedigrees and four individual patients, which were c.2722G>T, p.(Asp908Tyr), c.1657delA, p.(Ser553fs), c.2563_2564delCT, p.(Leu855fs), c.526T>G, p.(Leu176Val) as well as four previously reported mutations c. 3363_3364insT, p.(Lys1122fs), c. 2865_2866delGT, p.(Val955fs), c.1630C>T, p.(Arg544X), and c.2894C>T, p.(Pro965Leu). In silico analysis predicted that all the mutations reported were pathogenic. Limitations: We did not study how ADAR1 played its role in DSH. So, the exact pathogenic mechanism of ADAR1 in DSH patients wasn't clarified in this study. Conclusion: We found four novel ADAR1 mutations in this study. Our results enlarge the database on ADAR1 mutations associated with DSH.

ADAR1 up-regulates ZNF655 expression via RNA editing and enhances HBV replication in HepG2 cell line / 基础医学与临床

Objective To explore the effect of ADAR1 on ZNF655 and the regulation of ZNF655 on the expression of HBV. Methods Sanger sequencing was used to validate the 3′UTR region of ZNF655 in ADAR1. The expression of ADAR1 and ZNF655 mRNA as well as HBV RNA were detected by RT-qPCR. Dual luciferase report plasmid assay was used to detect the expression of luciferase. To detect the expression of ADAR1 and ZNF655 pro-tein by Western blot. HBsAg and HBeAg was detected by ELISA. Results The chr7:99575277 loci on ZNF655 3′UTR was homozygous in DNA level and hybrid in RNA level. On the 3′UTR editing site of ZNF655,the luciferase activity of the edited G allele was significantly higher than that of the normal A allele (P＜0.001). The expression of ZNF655 was upregulated by ADAR1 in the level of transcription and translation(P＜0.01).ZNF655 significantly promoted the expression of HBV. Conclusions The chr7:99575277 loci on ZNF655 3′UTR is edited by ADAR1, promoting the expression of ZNF655,which upregulated the expression of HBV.

Mutation analysis of the ADAR1 gene in a pedigree with dyschromatosis symmetrica hereditaria / 中华皮肤科杂志

Objective To detect mutations in the ARAD1 gene in a pedigree with dyschromatosis symmetrica hereditaria (DSH).Methods Genomic DNA was extracted from the peripheral blood of 8 family members (including 5 patients with DSH and 3 unaffected members) in the pedigree with DSH,as well as 100 unrelated healthy controls.All the 15 exon sequences of the ADAR1 gene were amplified by polymerase chain reaction (PCR)followed by direct sequencing.Then,mutations were detected in comparison with the standard sequence of the ADAR1 gene in Genebank.Results A nonsense mutation C.1420C ＞ T (p.Arg474X) was identified at position 1 420 in exon 2 of the ADAR1 gene in the 5 patients with DSH,but not in the 3 unaffected members or 100 unrelated healthy controls.Conclusion The nonsense mutation C.1420C ＞ T in the ADAR1 gene is the causative mutation in the pedigree with DSH.

ADAR1-mediated RNA-editing of 3'UTRs in breast cancer

BACKGROUND: Whole transcriptome RNA variant analyses have shown that adenosine deaminases acting on RNA ( ADAR ) enzymes modify a large proportion of cellular RNAs, contributing to transcriptome diversity and cancer evolution. Despite the advances in the understanding of ADAR function in breast cancer, ADAR RNA editing functional consequences are not fully addressed. RESULTS: We characterized A to G(I) mRNA editing in 81 breast cell lines, showing increased editing at 3'UTR and exonic regions in breast cancer cells compared to immortalized non-malignant cell lines. In addition, tumors from the BRCA TCGA cohort show a 24% increase in editing over normal breast samples when looking at 571 well-characterized UTRs targeted by ADAR1. Basal-like subtype breast cancer patients with high level of ADAR1 mRNA expression shows a worse clinical outcome and increased editing in their 3'UTRs. Interestingly, editing was particularly increased in the 3'UTRs of ATM, GINS4 and POLH transcripts in tumors, which correlated with their mRNA expression. We confirmed the role of ADAR1 in this regulation using a shRNA in a breast cancer cell line (ZR-75-1). CONCLUSIONS: Altogether, these results revealed a significant association between the mRNA editing in genes related to cancer-relevant pathways and clinical outcomes, suggesting an important role of ADAR1 expression and function in breast cancer.

The roles of RNA-editing enzyme ADAR1 in EV71 infection and virus mutation / 复旦学报（医学版）

Objective To identify the role of RNA-editing enzyme ADAR1 (adenosine deaminase acting on RNA) in EV71 infection and virus mutation.Methods RNAi technology was applied to establish ADAR1 knock-down stable cell lines.Then the cells were served to evaluate the role of ADAR1 in EV71 infection by MTT assay for detecting virus-induced cell viability,virus plaque assay for quantification of the virus titer and the cellular susceptibility to the virus,and Western blot for virus protein expressions.ADAR1-mediated RNA editing can result in the genetic A-G and T-C mutations.To further determine whether the effects of ADAR1 on EV71 infection were correlated with ADAR1-mediated EV71 RNA editing and therefore increased the viral mutations during the infection,the characteristics of EV71 mutation were analyzed based on the different full-length viral genomes from epidemic regions.The viral genome was also sequenced from the infected ADAR1 knock-down cells.Results After ADAR1 knock-down,the cell viability decreased quickly after the virus infection,and formed much more and larger sizes of plaques than the control cells.The virus capsid protein VP1 expressions and virus titer in the cells culture media were both increased in ADAR1 knockdown cells.Statistic analysis showed that A-G and T-C mutations were the major mutations of EV71,which were believed to be the hot sites for RNA-editing.However,the results of viral RNA genomic sequencing data indicated that ADAR1 did not edit EV71 genome directly.Conclusions ADAR1 was a restriction factor for controlling EV71.However,ADAR1 does not directly edit EV71 genome.

Adenosine to inosine acting on RNA enzymes 1 (ADAR1) contribute to the progression of tumor / 国际外科学杂志

Tumor occurrence and development is a complicated process.Previous studies confirm that DNA mutations result in activation of downstream pathways of tumor cells.Recently,studies of post-translational modifications showed that RNA editing play an important role in transcriptional mechanism.Thus,RNA editing further enrich the diversity of RNA and protein,participate in the process of many diseases,including cancer.Recently,many studies have focused on the RNA editing enzymne ADAR1 because of the complexity role in tumorigenesis and progression.This article mainly reviewed the role of ADAR1 in the occurrence and development of tumors.

The impact of RNA interference-induced ADAR1 down-regulation on cell proliferation of liver cancer / 中华肝胆外科杂志

Objective To observe the impact of RNA interference-induced ADAR1 down-regulation on cell proliferation of liver cancer.Methods Small interfering RNA (siRNA) was transfected into liver cancer cell line SMMC-7721.ADAR1 expression was detected by RT-PCR and Western blotting.Cell proliferation was determined by methyl thiazol tetrazolium (MTT) assay.Results After transfection for 24,48,and 72 h,ADAR1 mRNA expression was 0.612 ± 0.086,0.264 ± 0.018,0.156 ± 0.063 in experimental group and 1.032 ± 0.107,0.898 ± 0.092,0.968 ± 0.074 in control group,respectively.Experimental group had significantly lower ADAR1 mRNA than the other groups (P ＜ 0.05),and there was no statistically significant difference between control and blank group (P ＞ 0.05).ADAR1 protein relative expression was 0.684 ± 0.079,0.324 ± 0.042,0.145 ± 0.058 in experimental group and 1.002 ± 0.092,0.917 ± 0.068,0.972 ± 0.073 in control group,respectively,which was statistically significant (P ＜ 0.05).After transfection with siRNA,the proliferation ability of SMMC-7721 cells was enormously inhibited (P ＜ 0.05).Conclusion ADAR1 mRNA and protein level could be significantly decreased by specific RNA interference,and cell proliferation in SMMC-7721 cells were also greatly inhibited.

Effect of ADAR1 gene silencing in HaCaT cells on Wnt11 expression and tyrosinase activity in human A375 melanoma cells / 中华皮肤科杂志

Objective To estimate the influence of ADAR1 gene,which is considered to be responsible for the pathogenesis of dyschromatosis symmetrica hereditaria,on Wnt1 1 expression and tyrosinase activity.Methods Some cultured HaCaT cells were equally divided into four groups:control group remaining untreated,three experimental groups transfected with three different ADAR1-specific shRNAs respectively.Then,Western blot was performed to quantify the expression of Wnt11 protein in HaCaT cells so as to select the most potent shRNA.Some human A375 melanoma cells were cocultured with untransfected HaCaT cells (normally expressing ADAR1 and Wnt1 1 proteins) or HaCaT cells transfected with the selected specific shRNA (lowly expressing ADAR1 and Wnt11 proteins).Thereafter,cell appearance was observed using inverted microscopy at 24,48 and 72 hours,and tyrosinase activity was estimated at 48 hours.Results As Western blot showed,the expression of Wnt 11 protein was significantly lower in the three ADAR1-silenced experimental groups than in the control group.The number of dendritic protrusions at the junction sites between HaCaT cells and A375 cells was significantly decreased,together with a significant reduction in tyrosinase activity (absorbance value:0.0168 ± 0.0069 vs.0.0490 ± 0.0132,P ＜0.01),in A375 cells cocultured with transfected HaCaT cells compared with those cocultured with normal control HaCaT cells.Conclusion ADAR1 gene silencing in HaCaT cells can attenuate the expression of Wnt11 protein,and affect tyrosinase activity in A375 cells.