Editing independent effects of ADARs on the miRNA/siRNA pathways

Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR‐B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir‐376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase‐inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA‐binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions.     

ADAR1 is required for differentiation and neural induction by regulating microRNA processing in a catalytically independent manner

Adenosine deaminases acting on RNA (ADARs) are involved in adenosine-to-inosine RNA editing and are implicated in development and diseases. Here we observed that ADAR1 deficiency in human embryonic stem cells (hESCs) significantly affected hESC differentiation and neural induction with widespread changes in mRNA and miRNA expression, including upregulation of self-renewal-related miRNAs, such as miR302s. Global editing analyses revealed that ADAR1 editing activity contributes little to the altered miRNA/mRNA expression in ADAR1-deficient hESCs upon neural induction. Genome-wide iCLIP studies identified that ADAR1 binds directly to pri-miRNAs to interfere with miRNA processing by acting as an RNA-binding protein. Importantly, aberrant expression of miRNAs and phenotypes observed in ADAR1-depleted hESCs upon neural differentiation could be reversed by an enzymatically inactive ADAR1 mutant, but not by the RNA-binding-null ADAR1 mutant. These findings reveal that ADAR1, but not its editing activity, is critical for hESC differentiation and neural induction by regulating miRNA biogenesis via direct RNA interaction.     

Pin1 and WWP2 regulate GluR2 Q/R site RNA editing by ADAR2 with opposing effects

ADAR2 catalyses the deamination of adenosine to inosine at the GluR2 Q/R site in the pre-mRNA encoding the critical subunit of AMPA receptors. Among ADAR2 substrates this is the vital one as editing at this position is indispensable for normal brain function. However, the regulation of ADAR2 post-translationally remains to be elucidated. We demonstrate that the phosphorylation-dependent prolyl-isomerase Pin1 interacts with ADAR2 and is a positive regulator required for the nuclear localization and stability of ADAR2. Pin1(-/-) mouse embryonic fibroblasts show mislocalization of ADAR2 in the cytoplasm and reduced editing at the GluR2 Q/R and R/G sites. The E3 ubiquitin ligase WWP2 plays a negative role by binding to ADAR2 and catalysing its ubiquitination and subsequent degradation. Therefore, ADAR2 protein levels and catalytic activity are coordinately regulated in a positive manner by Pin1 and negatively by WWP2 and this may have downstream effects on the function of GluR2. Pin1 and WWP2 also regulate the large subunit of RNA Pol II, so these proteins may also coordinately regulate other key cellular proteins.     

Vero Cell Proteomic Changes Induced by Zika Virus Infection

The re‐emergence and the recent spread of the Zika virus (ZIKV) has raised significant global concerns due to lack of information in patient diagnosis and management. Thus, in addition to gaining more basic information about ZIKV biology, appropriate interventions and management strategies are being sought to control ZIKV‐associated diseases and its spread. This study's objective is to identify host cell proteins that are significantly dysregulated during ZIKV infection. SOMAScan, a novel aptamer‐based assay, is used to simultaneously screen >1300 host proteins to detect ZIKV‐induced host protein dysregulation at multiple time points during infection. A total of 125 Vero cell host proteins, including cytokines such as CXCL11 and CCL5, interferon stimulated gene 15, and translation initiation factors EIF5A and EIF4G2, are significantly dysregulated after ZIKV infection. Bioinformatic analyses of 77 host proteins, that are significantly dysregulated ≥1.25‐fold, identify several activated biological processes, including the JAK/STAT, Tec kinase, and complement cascade pathways.     

RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA

MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex.     

Solution structure of the N-terminal dsRBD of Drosophila ADAR and interaction studies with RNA

Adenosine deaminases that act on RNA (ADAR) catalyze adenosine to inosine (A-to-I) editing in double-stranded RNA (dsRNA) substrates. Inosine is read as guanosine by the translation machinery; therefore A-to-I editing events in coding sequences may result in recoding genetic information. Whereas vertebrates have two catalytically active enzymes, namely ADAR1 and ADAR2, Drosophila has a single ADAR protein (dADAR) related to ADAR2. The structural determinants controlling substrate recognition and editing of a specific adenosine within dsRNA substrates are only partially understood. Here, we report the solution structure of the N-terminal dsRNA binding domain (dsRBD) of dADAR and use NMR chemical shift perturbations to identify the protein surface involved in RNA binding. Additionally, we show that Drosophila ADAR edits the R/G site in the mammalian GluR-2 pre-mRNA which is naturally modified by both ADAR1 and ADAR2. We then constructed a model showing how dADAR dsRBD1 binds to the GluR-2 R/G stem-loop. This model revealed that most side chains interacting with the RNA sugar-phosphate backbone need only small displacement to adapt for dsRNA binding and are thus ready to bind to their dsRNA target. It also predicts that dADAR dsRBD1 would bind to dsRNA with less sequence specificity than dsRBDs of ADAR2. Altogether, this study gives new insights into dsRNA substrate recognition by Drosophila ADAR.     

Inhibition of hepatitis delta virus RNA editing by short inhibitory RNA-mediated knockdown of ADAR1 but not ADAR2 expression

Hepatitis delta virus (HDV) requires host RNA editing at the viral RNA amber/W site. Of the two host genes responsible for RNA editing via deamination of adenosines in double-stranded RNAs, short inhibitory RNA-mediated knockdown of host ADAR1 expression but not that of ADAR2 led to decreased HDV amber/W editing and virus production. Despite substantial sequence and structural variation among the amber/W sites of the three HDV genotypes, ADAR1a was primarily responsible for editing all three. We conclude that ADAR1 is primarily responsible for editing HDV RNA at the amber/W site during HDV infection.     

ADAR1介导的RNA编辑在血液肿瘤中的调控作用

RNA编辑是发生于双链RNA（dsRNA）上的一类重要转录后反应,可通过碱基插入、缺失或替换的方式改变RNA的核苷酸序列从而丰富转录组和蛋白质组水平的多样性。哺乳动物中最常见的RNA编辑是ADAR家族介导的腺嘌呤-次黄嘌呤编辑（A-to-I）,其在碱基配对过程中被识别为鸟嘌呤。人类转录组中已报道了数百万个A-to-I编辑位点,而ADAR1是最主要的催化酶。在血液肿瘤中,ADAR1的失调将直接影响基因编码区、非编码区和miRNA前体的A-to-I编辑状态,从而导致一系列分子事件改变,如蛋白质编码序列改变、内含子滞留、选择性剪接和miRNA生物发生受抑制。近年来研究发现,异常的RNA编辑导致分子调控网络的紊乱,促进细胞增殖、凋亡受阻和细胞耐药,是白血病干细胞（LSCs）生成和干性维持的重要因素。目前,以RNA编辑为靶点的新药（如rebecsinib）已经在动物实验中取得良好疗效。有别于传统抗肿瘤药,表观遗传抗肿瘤药有望克服血液肿瘤的耐药、复发难题,为患者提供全新治疗选择。本综述总结了ADAR1介导的RNA编辑在血液肿瘤中的作用机制及其生物学功能研究的进展,并探讨了其在药物研发和临床应用中的价值。     

Specificity of ADAR-mediated RNA editing in newly identified targets

Adenosine deaminases that act on RNA (ADARs) convert adenosines to inosine in both coding and noncoding double-stranded RNA. Deficiency in either ADAR1 or ADAR2 in mice is incompatible with normal life and development. While the ADAR2 knockout phenotype can be attributed to the lack of editing of the GluR-B receptor, the embryonic lethal phenotype caused by ADAR1 deficiency still awaits clarification. Recently, massive editing was observed in noncoding regions of mRNAs in mice and humans. Moreover, editing was observed in protein-coding regions of four mRNAs encoding FlnA, CyFip2, Blcap, and IGFBP7. Here, we investigate which of the two active mammalian ADAR enzymes is responsible for editing of these RNAs and whether any of them could possibly contribute to the phenotype observed in ADAR knockout mice. Editing of Blcap, FlnA, and some sites within B1 and B2 SINEs clearly depends on ADAR1, while other sites depend on ADAR2. Based on our data, substrate specificities can be further defined for ADAR1 and ADAR2. Future studies on the biological implications associated with a changed editing status of the studied ADAR targets will tell whether one of them turns out to be directly or indirectly responsible for the severe phenotype caused by ADAR1 deficiency.