Modulation of microRNA processing and expression through RNA editing by ADAR deaminases

Adenosine deaminases acting on RNA (ADARs) are involved in editing of adenosine residues to inosine in double-stranded RNA (dsRNA). Although this editing recodes and alters functions of several mammalian genes, its most common targets are noncoding repeat sequences, indicating the involvement of this editing system in currently unknown functions other than recoding of protein sequences. Here we show that specific adenosine residues of certain microRNA (miRNA) precursors are edited by ADAR1 and ADAR2. Editing of pri-miR-142, the precursor of miRNA-142, expressed in hematopoietic tissues, resulted in suppression of its processing by Drosha. The edited pri-miR-142 was degraded by Tudor-SN, a component of RISC and also a ribonuclease specific to inosine-containing dsRNAs. Consequently, mature miRNA-142 expression levels increased substantially in ADAR1 null or ADAR2 null mice. Our results demonstrate a new function of RNA editing in the control of miRNA biogenesis.     

Stress-induced apoptosis associated with null mutation of ADAR1 RNA editing deaminase gene

One type of RNA editing involves the conversion of adenosine residues into inosine in double-stranded RNA through the action of adenosine deaminases acting on RNA (ADAR). A-to-I RNA editing of the coding sequence could result in synthesis of proteins not directly encoded in the genome. ADAR edits also non-coding sequences of target RNAs, such as introns and 3'-untranslated regions, which may affect splicing, translation, and mRNA stability. Three mammalian ADAR gene family members (ADAR1-3) have been identified. Here we investigated phenotypes of mice homozygous for ADAR1 null mutation. Although live ADAR1-/- embryos with normal gross appearance could be recovered up to E11.5, widespread apoptosis was detected in many tissues. Fibroblasts derived from ADAR1-/- embryos were also prone to apoptosis induced by serum deprivation. Our results demonstrate an essential requirement for ADAR1 in embryogenesis and suggest that it functions to promote survival of numerous tissues by editing one or more double-stranded RNAs required for protection against stress-induced apoptosis.     

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.     

哺乳动物中作用于非编码RNA的RNA编辑研究进展

RNA编辑是RNA转录过程中序列变化而引起的一种基因动态调控机制。腺苷脱氨酶（adenosine deaminases acting on RNA, ADAR）参与RNA编辑,将双链RNA中腺苷残基（A）转化为肌苷（I）,接着被转录和拼接成鸟苷（G）。由ADAR催化,作用于RNA的A-I型RNA编辑是人类最常见的转录后修饰。近年来,这种修饰不仅存在于编码RNA中,在非编码RNA（noncoding RNA, ncRNA）中也逐渐被发现,如microRNA（miRNA）、小分子干扰RNA（siRNA）、转运RNA（tRNA）和长链非编码RNA（lncRNA）。这种修饰可能通过对microRNA和mRNA之间结合位点创造或破坏,进而影响ncRNA的生物起源、稳定性和靶向识别功能。目前,对这种生物现象的机制及ADAR底物,尤其是在ncRNA中的特性仍然没有得到充分的认识。主要对哺乳动物中ncRNA上的RNA编辑进行总结,并列举一些阐明其生物学功能的计算方法。     

ADAR1 RNA deaminase limits short interfering RNA efficacy in mammalian cells

Double-stranded RNA induces the homology-dependent degradation of cognate mRNA in the cytoplasm via RNA interference (RNAi) but also is a target for adenosine-to-inosine (A-to-I) RNA editing by adenosine deaminases acting on RNA (ADARs). An interaction between the RNAi and the RNA editing pathways in Caenorhabditis elegans has been suggested recently, but the precise mode of interaction remains to be established. In addition, it is unclear whether this interaction is possible in mammalian cells with their somewhat different RNAi pathways. Here we show that ADAR1 and ADAR2, but not ADAR3, avidly bind short interfering RNA (siRNA) without RNA editing. In particular, the cytoplasmic full-length isoform of ADAR1 has the highest affinity among known ADARs, with a subnanomolar dissociation constant. Gene silencing by siRNA is significantly more effective in mouse fibroblasts homozygous for an ADAR1 null mutation than in wild-type cells. In addition, suppression of RNAi effects are detected in fibroblast cells overexpressing functional ADAR1 but not when overexpressing mutant ADAR1 lacking double-stranded RNA-binding domains. These results identify ADAR1 as a cellular factor that limits the efficacy of siRNA in mammalian cells.     

Effects of conserved RNA secondary structures on hepatitis delta virus genotype I RNA editing, replication, and virus production

RNA editing of the hepatitis delta virus (HDV) antigenome at the amber/W site by the host RNA adenosine deaminase ADAR1 is a critical step in the HDV replication cycle. Editing is required for production of the viral protein hepatitis delta antigen long form (HDAg-L), which is necessary for viral particle production but can inhibit HDV RNA replication. The RNA secondary structural features in ADAR1 substrates are not completely defined, but base pairing in the 20-nucleotide (nt) region 3' of editing sites is thought to be important. The 25-nt region 3' of the HDV amber/W site in HDV genotype I RNA consists of a conserved secondary structure that is mostly base paired but also has asymmetric internal loops and single-base bulges. To understand the effect of this 3' region on the HDV replication cycle, mutations that either increase or decrease base pairing in this region were created and the effects of these changes on amber/W site editing, RNA replication, and virus production were studied. Increased base pairing, particularly in the region 15 to 25 nt 3' of the editing site, significantly increased editing; disruption of base pairing in this region had little effect. Increased editing resulted in a dramatic inhibition of HDV RNA synthesis, mostly due to excess HDAg-L production. Although virus production at early times was unaffected by this reduced RNA replication, at later times it was significantly reduced. Therefore, it appears that the conserved RNA secondary structure around the HDV genotype I amber/W site has been selected not for the highest editing efficiency but for optimal viral replication and secretion.     

RNA editing catalyzed by ADAR1 and its function in mammalian cells

In mammalian cells two active enzymes, ADAR1 and ADAR2, carry out A-to-I RNA editing. These two editases share many common features in their protein structures, catalytic activities, and substrate requirements. However, the phenotypes of the knockout animals are remarkably different, which indicate the distinct functions they play. The most striking effect of ADAR1 knockout is cell death and interruption of embryonic development that are not observed in ADAR2 knockout. Evidences have shown that ADAR1 plays critical roles in the differentiating cells in embryo and adult tissues to support the cell’s survival and permit their further differentiation and maturation. However, our knowledge in understanding of the mechanism by which ADAR1 exerts its unique effects is very limited. Many efforts had been made trying to understand why ADAR1 is so important that it is indispensible for animal survival, including studies that identify the RNA editing substrates and studies on non-editing mechanisms. The interest of this review is focused on the question why ADAR1 and not ADAR2 is required for cell survival. Therefore, only the data, published and unpublished, potentially connecting ADAR1 to its cell death effect is selectively cited and discussed here. The features of cell death caused by ADAR1 deletion are summarized. Potential involvement of interferon and protein kinase RNA-activated (PKR) pathways is proposed, but obviously more experimental evaluations are needed.     

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.