Scadden AD and O'Connell MA (2005), Cleavage of dsRNAs hyper-edited by ADARs occurs...

XB-ART-1176

Nucleic Acids Res 2005 Oct 27;3318:5954-64. doi: 10.1093/nar/gki909.

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Cleavage of dsRNAs hyper-edited by ADARs occurs at preferred editing sites.

Scadden AD , O'Connell MA .

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Long double-stranded RNAs (dsRNAs) may undergo covalent modification (hyper-editing) by adenosine deaminases that act on RNA (ADARs), whereby up to 50-60% of adenosine residues are converted to inosine. Previously, we have described a ribonuclease activity in various cell extracts that specifically targets dsRNAs hyper-edited by ADARs. Such a ribonuclease may play an important role in viral defense, or may alternatively be involved in down-regulation of other RNA duplexes. Cleavage of hyper-edited dsRNA occurs within sequences containing multiple IU pairs but not in duplexes that contain either isosteric GU pairs or Watson-Crick base pairs. Here, we describe experiments aimed at further characterizing cleavage of hyper-edited dsRNA. Using various inosine-containing dsRNAs we show that cleavage occurs preferentially at a site containing both IU and UI pairs, and that inclusion of even a single GU pair inhibits cleavage. We also show that cleavage occurs on both strands within a single dsRNA molecule and requires a 2'-OH group. Strikingly, we show that ADAR1, ADAR2 or dADAR all preferentially generate the preferred cleavage site when hyper-editing a long dsRNA.

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Species referenced: Xenopus laevis
Genes referenced: adar ag1 dut meox2 pam XB5937757

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	Figure 1. GU pairs have a deleterious effect on cleavage. (A) Cleavage assays were carried out using IIUI dsRNA molecules [5′ end-labeled on one strand ()] and substrates where IU pairs were replaced by GU pairs (Table 1). Time points used in these assays were 0, 0.5, 1 and 2 h. (B) Cleavage assays were carried out using control GU dsRNA molecules [5′ end-labeled on one strand ()], and substrates where GU pairs were replaced by IU pairs. Time points used in these assays were 0, 0.5, 1 and 2 h. (C) Data from cleavage assays as shown in (A) and (B) were quantitated following phosphorimaging (n ≥ 4). The amount of cleaved product is given as the percentage of the total amount of dsRNA. (D) Cleavage assays using dsRNA substrates [5′ end-labeled on one strand (*)] that contain four or five IU pairs were carried out. Time points used in these assays were 0, 0.5, 1 and 2 h. (E) Data from cleavage assays as shown in (D) were quantitated as described above (n ≥ 4). The amount of cleaved product is again given as the percentage of the total dsRNA. (F) A schematic diagram showing the position(s) of cleavage within the cleavage site sequence.
	Figure 2. 2′-OH groups are important for cleavage. (A) Cleavage assays were carried out using IIUI, dUT and dUB dsRNA molecules [5′ end-labeled on one strand (*)], to investigate the effect of dU residues on cleavage. Time points used in these assays were 0, 0.5, 1 and 2 h. (B) Data from cleavage assays as shown in (A) were quantitated following phosphor imaging (n ≥ 4). The amount of cleaved product is given as the percentage of the total dsRNA.
	Figure 3. Cleavage of IIUI dsRNA occurs on both strands within a single molecule. (A) Cleavage assays were carried out using IIUI or GU hairpins (Table 1). The GU hairpin was 5′ end-labeled () while the IIUI hairpin was labeled either on the 5′ end () or internally, adjacent to the tetraloop sequence (*). Time points used in this assay were 0, 0.5, 1 and 2 h. Positions of cleavage within the 5′ end-labeled or internally labeled IIUI hairpins are shown schematically in (B) and (C), respectively. The product corresponding to cleavage on the top strand of the 5′ end-labeled IIUI hairpin is indicated by an open square while cleavage on the bottom strand is indicated by a closed square. Cleavage products corresponding to cleavage on either the top or the bottom strand of the internally labeled IIUI hairpin are indicated by an open circle (∼34–37 nt), while cleavage products corresponding to cleavage on both strands are indicated by a closed circle (∼24 nt). Molecular weight markers (nt) are shown at the left of the figure. (B) Cleavage of the 5′ end-labeled IIUI hairpin is shown schematically. The blue line represents the 32P-labeled cleaved product(s) following cleavage at the site(s) indicated by the arrow(s). The striped line indicates cleavage at multiple positions on the bottom strand. (C) Cleavage of the internally labeled IIUI hairpin is shown schematically. The pink line represents the 32P-labeled cleaved product(s) following cleavage at the site(s) indicated by the arrow(s). The striped line indicates cleavage at multiple positions on the bottom strand.
	Figure 4. Cleavage occurs preferentially between the IU and UI pair in the IIUI sequence. (A) Cleavage assays were carried out using IIUI and IIUI-pal dsRNA molecules [5′ end-labeled on one strand (*)]. Time points used in these assays were 0, 0.5, 1 and 2 h. (B) Data from cleavage assays as shown in (A) were quantitated following phosphor imaging (n ≥ 4). The amount of cleaved product is given as the percentage of the total amount of dsRNA. (C) A schematic diagram showing the position(s) of cleavage within the IIUI and IIUI-pal dsRNAs.
	Figure 5. IIUI is better than the 4I site for cleavage. (A) Sequence of IIUI and COMP dsRNAs. The two cleavage sites in the COMP dsRNA are labeled as IIUI (open circle) and 4I (closed circle). (B) Cleavage assays were carried out using IIUI and COMP dsRNA molecules [5′ end-labeled on one strand (*)]. Time points used in these assays were 0, 0.5, 1 and 2 h. Cleavage on the top strand of the COMP dsRNA at the 4I site is indicated by a closed circle, while cleavage at the IIUI site on either strand of the COMP dsRNA is indicated by an open circle. (C) Data from cleavage assays as shown in (A) were quantified following phosphorimaging (n ≥ 4). The amount of cleaved product is given as the percentage of the total amount of dsRNA. Cleavage of COMP dsRNA occurred at the IIUI sequence (COMP IIUI) or at the 4I sequence (COMP 4I).
	Figure 6. Editing by ADAR1, ADAR2 or dADAR generates the IIUI sequence. (A) Sequence of ΔKP RNA (sense strand). An example of an adenosine triplet (A217–219) is shown in bold. The cleavage site sequence (A200–203) is underlined. (B) Efficiency of editing of ΔKP by ADAR2. Efficiency of editing is expressed as the percentage of clones that contain an inosine residue at each edited position. Hundred percent editing is indicated by a dotted line. Editing of the antisense and sense strands are shown at the top and bottom of the graph, respectively. Red bars show an example of editing of an adenosine triplet (A200–203). (C) The average editing (%) of each adenosine preceded by a particular nucleotide (AAX, CAX, GAX, UAX) was calculated to give 5′ neighbor preferences. Similarly, the average editing (%) of each adenosine followed by a particular nucleotide (XAA, XAC, XAG, XAU) was calculated to give 3′ neighbor preferences. The total number of adenosines in each context is shown in brackets. (D) Efficiency of editing of ΔKP (antisense strand) by ADAR2, ADAR1 or dADAR. Efficiency of editing is expressed as the percentage of clones that contain an inosine residue at each edited position. Hundred percent editing is indicated by a dotted line. Blue bars indicate particular adenosine residues differentially edited by the three ADARs. The green bar indicates the adenosine residue within the cleavage site sequence.

References [+] :

Bass, RNA editing by adenosine deaminases that act on RNA. 2002, Pubmed