Specific cleavage of hyper-edited dsRNAs - PubMed (original) (raw)
Specific cleavage of hyper-edited dsRNAs
A D Scadden et al. EMBO J. 2001.
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.
Figures
Fig. 1. (A) Quantitation of deamination. ΔKP dsRNA was deaminated to varying degrees using ADAR2. Digestion of the modified RNA using RNase P1, followed by TLC, enabled quantitation of the amount of A to I conversion. The RNAs used in the assays shown typically contained ∼40% A to I conversion (lane 8). (B) Deamination of ΔKP results in a homogenous population of RNA. As the level of deamination of ΔKP increased, there was a corresponding reduction in the mobility of the RNA on a native gel (lanes 1–8). For dsRNAs with ∼40% A to I conversion (lane 8), the RNA migrated as a relatively compact band (‘d-ds’), indicating that all RNAs in the population were modified. (C) Deaminated dsRNA is specifically cleaved. Incubation of ΔKP d-dsRNA in Xenopus oocyte extract gave rise to two discrete cleavage products (lanes 1–4). In contrast, ΔKP dsRNA was stable (lanes 5–8), and ΔKP ssRNA was degraded slowly yielding numerous products (lanes 9–12). The time course used in each assay was 0, 15, 60 and 90 min. Positions of DNA molecular weight markers are shown to the right of the figure (φX174 _Hae_III, 310, 281, 271, 234, 194, 118, and 72 nt). (D) Incubation of both PV and CAT d-dsRNAs for 90 min in Xenopus oocyte extracts also gave rise to discrete cleavage products (lanes 1–6 and 7–12, respectively). The equivalent dsRNAs were stable (compare lanes 2 and 4, and lanes 8 and 10), while degradation of the ssRNAs gave more numerous products (lanes 6 and 12).
Fig. 2. (A) d-dsRNA is cleaved by a cytoplasmic ribonuclease. ΔKP d-dsRNA was specifically cleaved to give two major products when incubated in either Xenopus oocyte extract or HeLa cell S100 (lanes 1–4 and 13–16, respectively). In contrast, ΔKP d-dsRNA was predominantly stable in HeLa cell nuclear extract (NE; lanes 5–8). The minor amount of cleavage detected could probably be accounted for by cytoplasmic contamination of the nuclear extract. ΔKP dsRNA was stable in both HeLa cell S100 and NE extracts (lanes 17–20 and 9–12, respectively). A cytoplasmic activity thus appears to be responsible for cleavage of d-dsRNA. The time course used in each assay was 0, 15, 60 and 90 min. (B) ΔKP d-dsRNA does not undergo photocleavage. Cleavage of ΔKP d-dsRNA in Xenopus oocyte extract was identical in the light and dark (compare lanes 1–3 and 4–6, respectively). This indicated that photocleavage was not responsible for the observed cleavage. The time course used in each assay was 0, 30 and 60 min.
Fig. 3. (A) Identification of the sites of deamination in ΔKP d-dsRNA. Cloned RT–PCR products derived from a ΔKP d-dsRNA template deaminated to 40% were sequenced to identify positions of deamination. The sequence shown corresponds to the sense strand of ΔKP, and positions of A to I conversion are indicated by ‘I’. This sequence is typical of a number of sequenced clones. Cleavage of the d-dsRNA occurred at the sequence IIUI (boxed; referred to as CS). The sequence underlined (IIII) is referred to as 4I. The double-stranded sequence of the CS and 4I sites is indicated. (B) Two mutants of ΔKP were generated, where mutations were made in either the CS (ΔU mutant) or 4I (+U mutant) sequences, as shown (sense strand). ΔU potentially contained no cleavage sites while +U potentially contained two cleavage sites (compared with the single site in wild-type (WT) ΔKP). (C) Alternating IU and UI base pairs results in cleavage. ΔKP d-dsRNA (WT) was cleaved at the sequence CS in Xenopus oocyte extract to give two products (lane 14). In contrast, incubation of ΔU d-dsRNA yielded no cleavage products (lanes 1–4). Cleavage of +U d-dsRNA resulted in several products that corresponded to cleavage at both the CS and modified 4I sequences (lanes 7–10). The equivalent WT, ΔU and +U dsRNAs were stable (lanes 16, 6 and 12). The time course used to assay ΔU and +U d-dsRNAs was 0, 15, 60 and 90 min. (D) Mutants of ΔKP were generated where the WT CS sequence (AAUA) was replaced by the given sequences. The corresponding d-dsRNAs were incubated in Xenopus oocyte extract, and the cleavage was analysed by phosphoimaging. The initial rate of cleavage for each mutant (fmol full-length RNA cleaved/min) was calculated, as given in the table. The graph shows the cleavage of each d-dsRNA over a 90 min time course. These data indicate that sequences which potentially contain different arrangements of IU and UI base pairs are not cleaved with equal efficiently following hyper-editing.
Fig. 4. (A) Cleavage of short synthetic dsRNA substrates. Short synthetic dsRNAs that contained the CS sequence were used to analyse cleavage. Incubation of short dsRNAs, 5′ end-labelled on either the sense (S) or antisense strand (AS), in Xenopus oocyte extract indicated that both RNA strands were cleaved (lanes 3 and 6). Electrophoresis alongside an alkaline hydrolysis ladder (H; lanes 1 and 4) enabled mapping of the cleavage sites on the sense and antisense strands. (B) Mapping the cleavage sites. The short synthetic RNAs were cleaved at the positions indicated. The sense strand was cleaved at one major position, 5′ of a U residue, and within the sequence of alternating IU and UI base pairs. In contrast, the antisense strand was cleaved at five positions, where ∼90% of the cleavage occurred 5′ of U residues. (C) Alternating GU and UG base pairs are not cleaved. ΔKP dsRNA was synthesized where the sequence of the cleavage site comprised GU and UG base pairs (as indicated above gel). In contrast with ΔKP d-dsRNA, this dsRNA was not cleaved when incubated in Xenopus oocyte extract (compare lanes 1–4 and 5–8). The time course used in each assay was 0, 15, 60 and 90 min. (D) Short dsRNAs that contained either alternating IU and UI base pairs or GU and UG base pairs (indicated above gel) were incubated in Xenopus oocyte extract. The time course used in each assay was 0, 30, 60 and 120 min. Each of the RNAs shown is labelled on the sense strand (indicated by *). While the dsRNAs containing alternating IU and UI base pairs were cleaved (lanes 1–4), the dsRNAs that contained GU and UG base pairs were stable (lanes 5–8). Thus, substitution of guanosine residues for inosine residues in the cleavage site inhibits cleavage of the RNA. (E) A non-Watson–Crick GU wobble base pair. An IU wobble base pair is isosteric with the GU base pair, but lacks the exocyclic amine group (circled) which projects into the minor groove.
References
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