Article

Specific cleavage of hyper-edited dsRNAs

Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK
The EMBO Journal (Impact Factor: 10.75). 09/2001; 20(15):4243-52. DOI: 10.1093/emboj/20.15.4243
Source: PubMed

ABSTRACT Extended double-stranded DNA (dsRNA) duplexes can be hyper-edited by adenosine deaminases that act on RNA (ADARs). Long uninterrupted dsRNA is relatively uncommon in cells, and is frequently associated with infection by DNA or RNA viruses. Moreover, extensive adenosine to inosine editing has been reported for various viruses. A number of cellular antiviral defence strategies are stimulated by dsRNA. An additional mechanism to remove dsRNA from cells may involve hyper-editing of dsRNA by ADARs, followed by targeted cleavage. We describe here a cytoplasmic endonuclease activity that specifically cleaves hyper-edited dsRNA. Cleavage occurs at specific sites consisting of alternating IU and UI base pairs. In contrast, unmodified dsRNA and even deaminated dsRNAs that contain four consecutive IU base pairs are not cleaved. Moreover, dsRNAs in which alternating IU and UI base pairs are replaced by isomorphic GU and UG base pairs are not cleaved. Thus, the cleavage of deaminated dsRNA appears to require an RNA structure that is unique to hyper-edited RNA, providing a molecular target for the disposal of hyper-edited viral RNA.

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    • "It was suggested that inosine-containing transcripts are subject to specific regulation, affecting the fate of the RNA molecule (Rueter et al. 1999; Scadden and Smith 2001; Zhang and Carmichael 2001; Prasanth et al. 2005; Liang and Landweber 2007; Scadden 2007). However, given the wide scope of Alu editing in the human transcriptome , a large fraction of human transcripts contains inosines, suggesting that additional factors must be involved in these RNA regulations. "
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    ABSTRACT: RNA molecules transmit the information encoded in the genome and generally reflect its content. Adenosine-to-inosine (A-to-I) RNA editing by ADAR proteins converts a genomically encoded adenosine into inosine. It is known that most RNA editing in human takes place in the primate-specific Alu sequences, but the extent of this phenomenon and its effect on transcriptome diversity is not yet clear. Here, we analyzed large-scale RNA-seq data and detected over 1.6 million editing sites. As detection sensitivity increases with sequencing coverage, we performed ultra-deep sequencing of selected Alu sequences and showed that the scope of editing is much larger than anticipated. We found that virtually all adenosines within Alu repeats that form double-stranded RNA undergo A-to-I editing, although most sites exhibit editing at only low levels (<1%). Moreover, using high coverage sequencing, we observed editing of transcripts resulting from residual anti-sense expression, doubling the number of edited sites in the human genome. Based on bioinformatic analyses and deep targeted sequencing, we estimate that there are over 100 million human Alu RNA editing sites, located in the majority of human genes. These findings set the stage for exploring how this primate-specific massive diversification of the transcriptome is utilized.
    Genome Research 12/2013; 24(3). DOI:10.1101/gr.164749.113 · 13.85 Impact Factor
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    • "Finally, A-to-I editing may target RNAs for degradation (Fig. 3). A ribonuclease was identified that specifically targeted hyper-edited duplex RNAs, with efficient cleavage within a region of multiple I:U base pairs (Scadden and Smith, 2001b). ADAR1 and ADAR2 generated the cleavage site when hyper-editing long dsRNA (Scadden and O'Connell, 2005). "
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    ABSTRACT: A-to-I RNA editing, the deamination of adenosine (A) to inosine (I) that occurs in regions of RNA with double-stranded character, is catalyzed by a family of Adenosine Deaminases Acting on RNA (ADARs). In mammals there are three ADAR genes. Two encode proteins that possess demonstrated deaminase activity: ADAR1, which is interferon-inducible, and ADAR2 which is constitutively expressed. ADAR3, by contrast, has not yet been shown to be an active enzyme. The specificity of the ADAR1 and ADAR2 deaminases ranges from highly site-selective to non-selective, dependent on the duplex structure of the substrate RNA. A-to-I editing is a form of nucleotide substitution editing, because I is decoded as guanosine (G) instead of A by ribosomes during translation and by polymerases during RNA-dependent RNA replication. Additionally, A-to-I editing can alter RNA structure stability as I:U mismatches are less stable than A:U base pairs. Both viral and cellular RNAs are edited by ADARs. A-to-I editing is of broad physiologic significance. Among the outcomes of A-to-I editing are biochemical changes that affect how viruses interact with their hosts, changes that can lead to either enhanced or reduced virus growth and persistence depending upon the specific virus.
    Virology 03/2011; 411(2):180-93. DOI:10.1016/j.virol.2010.12.004 · 3.28 Impact Factor
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    • "This is supported by the abundance of hyperediting clusters in splicing-defective transcripts (Kim et al. 2004). This idea is in line with a similar proposed mechanism, suggesting that an I-specific cleavage of RNAs can lead to the selective destruction of edited RNAs (Scadden and Smith 2001). "
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    ABSTRACT: A-to-I RNA editing is the conversion of adenosine to inosine in double-stranded cellular and viral RNAs. Recently, abundant hyperediting of human transcripts, affecting thousands of genes, has been reported. Most of these editing sites are confined to intramolecular hairpin double-stranded RNA (dsRNA) structures formed by pairing of neighboring, reversely oriented, primate-specific Alu repeats. The biological implication of this extensive modification is still a mystery. A number of studies have shown that heavily edited transcripts are often retained in the nucleus. A recent study found that the edited region in transcripts of the mouse Slc7a2 gene is post-transcriptionally cleaved upon stress, enabling the release of the mRNA to the cytoplasm, followed by its translation. Here, we aim to test whether this scenario might be relevant for many other hyperedited Alu targets. Bioinformatics analysis of publicly available mRNA and expressed sequence tag data provides evidence showing that neighboring, reversely oriented, Alu elements are often cleaved at both ends of the region harboring the inverted repeats followed by rejoining of the two parts of the transcript on both sides of the inverted repeats, resulting in almost inosine-free mRNA products. Deleted segments vary among transcripts of the same gene and are not flanked by the canonical splicing signal sequences. The tissue distribution of these events seems to correlate with known A-to-I editing patterns, suggesting that it depends on the dsRNA structure being edited. Results are experimentally verified by polymerase chain reaction and cloning data. A database of 566 human and 107 mouse putative cleavage loci is supplied.
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