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PubMed2005

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.

Adenosine Deaminase↗
PubMed2005

High doses of siRNAs induce eri-1 and adar-1 gene expression and reduce the efficiency of RNA interference in the mouse.

RNAi (RNA interference) is a gene-silencing mechanism that is conserved in evolution from worm to human and has been a powerful tool for gene functional research. It has been clear that the RNAi effect triggered by endogenous or exogenous siRNAs (small interfering RNAs) is transient and dose-dependent. However, there is little information on the regulation of RNAi. Recently, some proteins that regulate the RNA-silencing machinery have been identified. We have observed in previous work that the expression of target genes rebounds after being suppressed for a period of time by siRNAs. In the present study, we used secretory hepatitis B virus surface antigen gene as a reporter and compared its expression level in cell culture and mice challenged by different doses of siRNAs. A quicker and higher rebound of gene expression was observed in mice tail-vein-injected with higher doses of siRNA, and the rebound was associated with an increase in the mRNA level of meri-1 (mouse enhanced RNAi) and adar-1 (adenosine deaminase acting on RNA) genes encoding an exonuclease and RNA-specific adenosine deaminase respectively. Down-regulation of meri-1 by RNAi enhanced the sensitivity and efficiency of siRNA in inhibiting the expression of hepatitis B virus surface antigen. These results indicate that RNAi machinery may be under negative regulation, through the induction of a series of genes coding for destabilizing enzymes, by siRNAs introduced into the cell, and also suggest that a suitable amount of siRNA should be used for research or therapeutic applications.

Adenosine Deaminase↗
PubMed2001

Aromatic di-alanine repeats (AdAR) are structural motifs characteristic of the soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) family.

The aromatic di-alanine repeat is a novel 12-amino acid-long motif constituting alternate small and large hydrophobic residues that mediate the close packing of alpha-helices. A hidden Markov model profile was constructed from the motifs initially described in Soluble N-ethyl maleimide-sensitive factor attachment proteins (SNAP), a family of soluble proteins involved in intracellular membrane fusion. Scanning different sets of protein sequences showed unambiguously that this profile defines a structural motif independent of the tetratrico peptide repeat, another widespread alpha-helical motif. In addition to SNAP, aromatic di-alanine repeats are found in selective LIM homeodomain binding proteins (SLB) and in proteins from the Pyrococcus and Archaeoglobus prokaryotes.

Alanine↗
PubMed2005

Heterochromatin: on the ADAR radar?

Vigilin proteins, the absence of which is known to cause abnormalities in heterochromatin, have been found to bind edited RNAs. Molecular complexes including vigilin comprise proteins involved with RNA editing and with DNA repair, making connections between these processes and RNA-based silencing mechanisms.

Autoantigens↗
PubMed2002

The promoter-proximal KCS element of the PKR kinase gene enhances transcription irrespective of orientation and position relative to the ISRE element and is functionally distinct from the KCS-like element of the ADAR deaminase Promoter.

The RNA-dependent protein kinase PKR promoter is interferon (IFN) inducible and possesses a novel 15-base pair (bp) constitutive activator element, designated kinase conserved sequence (KCS), in addition to an IFN-stimulated response element (ISRE). Deletion of the KCS element or point mutations within the KCS element greatly reduce both basal and IFN-inducible PKR promoter activity. The IFN-inducible RNA-specific adenosine deaminase ADAR1 promoter possesses a KCS-like (KCS-l) element. The sequences of the KCS and KCS-l elements and their positions relative to the cognate ISRE element are similar between the PKR and ADAR1 promoters. However, substitution of the ADAR1 KCS-l element for the KCS element of the PKR promoter resulted in significantly reduced basal and IFN-inducible promoter activities comparable to either point mutation or entire deletion of the PKR KCS element. The PKR KCS element selectively bound nuclear proteins more efficiently than did the ADAR1 KCS-l element. Reversing the positions of the KCS and ISRE elements of the PKR promoter relative to one another or reversing the orientation of either element while conserving the naturally occurring 4-bp spacing between the two elements did not significantly reduce basal or IFN-inducible promoter activity. Taken together, these results are consistent with the notion that the KCS and ISRE elements of the PKR promoter function as a unit.

Adenosine Deaminase↗
PubMed1999

Caenorhabditis elegans mRNAs that encode a protein similar to ADARs derive from an operon containing six genes.

The Caenorhabditis elegans T20H4.4 open reading frame (GenBank accession no. U00037) predicted by Genefinder encodes a 367 amino acid protein that is 32-35% identical to the C-terminal domain of adenosine deaminases that act on RNA. We show that T20H4.4 cDNAs (GenBank accession no. AF051275) encode a larger 495 amino acid protein that is extended at its N-terminus to include a single double-stranded RNA-binding motif, and that T20H4.4 occupies the second position in a six-gene operon (5'-T20H4.5, T20H4.4, R151.8A, R151.8B, R151.7, R151.6-3'). Ten different spliced-leader (SL) sequences were found attached to T20H4.4 mRNAs, including SL1, SL2 and eight SL2-like leaders that include two new variants. Characterization of cDNAs derived from all six genes confirmed the essential features of C.elegans operons: intercistronic distances in the range of 104-257 nt between the upstream polyadenylation sites and the downstream trans -splice sites; SL2, or SL2-like leaders, attached to the downstream mRNAs. Polycistronic mRNA fragments revealed a 5'-untranslated region (5'-UTR) >705 nt. The 5'-UTR is removed in mature mRNAs from the first gene (T20H4.5) and replaced primarily by SL1, and to a lesser extent by SL2. Our study provides new information regarding operons and how they are processed.

5' Untranslated Regions↗
PubMed2006

The large form of ADAR 1 is responsible for enhanced hepatitis delta virus RNA editing in interferon-alpha-stimulated host cells.

Hepatitis delta virus (HDV) RNA editing controls the formation of hepatitis-delta-antigen-S and -L and therefore indirectly regulates HDV replication. Editing is thought to be catalysed by the adenosine deaminase acting on RNA1 (ADAR1) of which two different forms exist, interferon (IFN)-alpha-inducible ADAR1-L and constitutively expressed ADAR1-S. ADAR1-L is hypothesized to be a part of the innate cellular immune system, responsible for deaminating adenosines in viral dsRNAs. We examined the influence of both forms on HDV RNA editing in IFN-alpha-stimulated and unstimulated hepatoma cells. For gene silencing, an antisense oligodeoxyribonucleotide against a common sequence of both forms of ADAR1 and another one specific for ADAR1-L alone were used. IFN-alpha treatment of host cells led to approximately twofold increase of RNA editing compared with unstimulated controls. If ADAR1-L expression was inhibited, this substantial increase in editing could no longer be observed. In unstimulated cells, ADAR1-L suppression had only minor effects on editing. Inhibition of both forms of ADAR1 simultaneously led to a substantial decrease of edited RNA independently of IFN-alpha-stimulation. In conclusion, the two forms of ADAR1 are responsible almost alone for HDV editing. In unstimulated cells, ADAR1-S is the main editing activity. The increase of edited RNA under IFN-alpha-stimulation is because of induction of ADAR1-L, showing for the first time that this IFN-inducible protein is involved in the base modification of replicating HDV RNA. Thus, induction of ADAR1-L may at least partially cause the antiviral effect of IFN-alpha in natural immune response to HDV as well as in case of therapeutic administration of IFN.

Adenosine Deaminase↗
PubMed1997

Functionally distinct double-stranded RNA-binding domains associated with alternative splice site variants of the interferon-inducible double-stranded RNA-specific adenosine deaminase.

The double-stranded RNA-specific adenosine deaminase (ADAR) is an interferon-inducible RNA-editing enzyme implicated in the site-selective deamination of adenosine to inosine in viral RNAs and cellular pre-mRNAs. We have isolated and characterized human genomic clones of the ADAR gene and cDNA clones encoding splice site variants of the ADAR protein. Southern blot and sequence analyses revealed that the gene spans about 30 kilobase pairs and consists of 15 exons. The codon phasing of the splice site junctions of exons 3, 5, and 7 that encode the three copies of the highly conserved RNA-binding R-motif (RI, RII, and RIII) was exactly conserved and identical to those R-motif exons of the interferon-inducible RNA-dependent protein kinase. Alternative splice site variants of the 1226-amino acid ADAR-a protein, designated b and c, were identified that differed in exons 6 and 7. ADAR-b was a 5'-splice site variant that possessed a 26-amino acid deletion within exon 7; ADAR-c was a 3'-splice site variant that possessed an additional 19-amino acid deletion within exon 6. The wild-type ADAR-a, -b, and -c proteins all possessed comparable double-stranded RNA-specific adenosine deaminase activity. However, mutational analysis of the R-motifs revealed that the exon 6 and 7 deletions of ADAR-b and -c variants altered the functional importance of each of the three R-motifs.

Adenosine Deaminase↗
PubMed1998

Double-stranded RNA-specific adenosine deaminase: nucleic acid binding properties.

The RNA-specific adenosine deaminase (ADAR1, herein referred to as ADAR) is an interferon-inducible RNA-editing enzyme. ADAR catalyzes the C-6 deamination of adenosine in double-stranded (ds) structures present in viral RNAs and cellular pre-mRNAs as well as synthetic dsRNA substrates. ADAR possesses three functionally distinct copies of the highly conserved double-stranded RNA binding R motif (RI, RII, RIII) implicated in the recognition of dsRNA structures within the substrate RNAs. ADAR is also a Z-DNA-binding protein. Two Z-DNA binding motifs (Zalpha and Zbeta) present in ADAR correspond to repeated regions homologous to the N-terminal region of the vaccinia virus E3L protein. Here we describe assay methods for measurement of ADAR enzymatic activity, dsRNA binding activity, and Z-DNA binding activity.

Adenosine Deaminase↗
PubMed2002

RNA hairpins in noncoding regions of human brain and Caenorhabditis elegans mRNA are edited by adenosine deaminases that act on RNA.

Adenosine deaminases that act on RNA (ADARs) constitute a family of RNA-editing enzymes that convert adenosine to inosine within double-stranded regions of RNA. We previously developed a method to identify inosine-containing RNAs and used it to identify five ADAR substrates in Caenorhabditis elegans. Here we use the same method to identify five additional C. elegans substrates, including three mRNAs that encode proteins known to affect neuronal functions. All 10 of the C. elegans substrates are edited in long stem-loop structures located in noncoding regions, and thus contrast with previously identified substrates of other organisms, in which ADARs target codons. To determine whether editing in noncoding regions was a conserved ADAR function, we applied our method to poly(A)+ RNA of human brain and identified 19 previously unknown ADAR substrates. The substrates were strikingly similar to those observed in C. elegans, since editing was confined to 3' untranslated regions, introns, and a noncoding RNA. Also similar to what was found in C. elegans, 15 of the 19 substrates were edited in repetitive elements. The identities of the newly identified ADAR substrates suggest that RNA editing may influence many biologically important processes, and that for many metazoa, A-to-I conversion in coding regions may be the exception rather than the rule.

3' Untranslated Regions↗
PubMed2003

Requirement of dimerization for RNA editing activity of adenosine deaminases acting on RNA.

Adenosine deaminases acting on RNA (ADAR) convert adenosine residues into inosines in double-stranded RNA. Three vertebrate ADAR gene family members, ADAR1, ADAR2, and ADAR3, have been identified. The catalytic domain of all three ADAR gene family members is very similar to that of Escherichia coli cytidine deaminase and APOBEC-1. Homodimerization is essential for the enzyme activity of those cytidine deaminases. In this study, we investigated the formation of complexes between differentially epitope-tagged ADAR monomers by sequential affinity chromatography and size exclusion column chromatography. Both ADAR1 and ADAR2 form a stable enzymatically active homodimer complex, whereas ADAR3 remains as a monomeric, enzymatically inactive form. No heterodimer complex formation among different ADAR gene family members was detected. Analysis of HeLa and mouse brain nuclear extracts suggested that endogenous ADAR1 and ADAR2 both form a homodimer complex. Interestingly, endogenous ADAR3 also appears to form a homodimer complex, indicating the presence of a brain-specific mechanism for ADAR3 dimerization. Homodimer formation may be necessary for ADAR to act as active deaminases. Analysis of dimer complexes consisting of one wild-type and one mutant monomer suggests functional interactions between the two subunits during site-selective RNA editing.

Adenosine Deaminase↗
PubMed2002

Increased RNA editing and inhibition of hepatitis delta virus replication by high-level expression of ADAR1 and ADAR2.

Hepatitis delta virus (HDV) is a subviral human pathogen that uses specific RNA editing activity of the host to produce two essential forms of the sole viral protein, hepatitis delta antigen (HDAg). Editing at the amber/W site of HDV antigenomic RNA leads to the production of the longer form (HDAg-L), which is required for RNA packaging but which is a potent trans-dominant inhibitor of HDV RNA replication. Editing in infected cells is thought to be catalyzed by one or more of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). We examined the effects of increased ADAR1 and ADAR2 expression on HDV RNA editing and replication in transfected Huh7 cells. We found that both ADARs dramatically increased RNA editing, which was correlated with strong inhibition of HDV RNA replication. While increased HDAg-L production was the primary mechanism of inhibition, we observed at least two additional means by which ADARs can suppress HDV replication. High-level expression of both ADAR1 and ADAR2 led to extensive hyperediting at non-amber/W sites and subsequent production of HDAg variants that acted as trans-dominant inhibitors of HDV RNA replication. Moreover, we also observed weak inhibition of HDV RNA replication by mutated forms of ADARs defective for deaminase activity. Our results indicate that HDV requires highly regulated and selective editing and that the level of ADAR expression can play an important role: overexpression of ADARs inhibits HDV RNA replication and compromises virus viability.

Adenosine Deaminase↗