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Specific cleavage of hyper-edited dsRNAs

2001; Springer Nature; Volume: 20; Issue: 15 Linguagem: Inglês

10.1093/emboj/20.15.4243

1460-2075

A.D.J. Scadden, Christopher W. J. Smith,

Herpesvirus Infections and Treatments

Article1 August 2001free access Specific cleavage of hyper-edited dsRNAs A.D.J. Scadden A.D.J. Scadden Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1GA UK Search for more papers by this author Christopher W.J. Smith Corresponding Author Christopher W.J. Smith Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1GA UK Search for more papers by this author A.D.J. Scadden A.D.J. Scadden Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1GA UK Search for more papers by this author Christopher W.J. Smith Corresponding Author Christopher W.J. Smith Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1GA UK Search for more papers by this author Author Information A.D.J. Scadden1 and Christopher W.J. Smith 1 1Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1GA UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:4243-4252https://doi.org/10.1093/emboj/20.15.4243 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Eukaryotic cells possess a number of sensitive mechanisms to detect and respond to the presence of extended double-stranded RNA (dsRNA) (Maquat and Carmichael, 2001). While short dsRNA duplexes (<10 bp) play important roles in a variety of cellular processes, there are relatively few examples of long uninterrupted dsRNA duplexes in cells. One source is the presence of antisense RNA transcripts, which have been implicated in the regulation of gene expression by forming duplexes with the sense strand mRNA (reviewed by Kumar and Carmichael, 1998). However, the presence of dsRNA is more commonly indicative of viral infection or of the presence of other invading nucleic acid elements. The presence of dsRNA in mammalian cells is sufficient to elicit an antiviral response mediated by enzymes such as dsRNA-dependent protein kinase (PKR) (Samuel, 1979, 1998) or 2′-5′-oligoadenylate synthetase/RNase L (Zhou et al., 1993; Silverman et al., 1997), which mediate global down-regulation of translation and mRNA stability, respectively. In contrast, the more recently characterized phenomenon of RNA interference (RNAi) involves targeted post-transcriptional repression only of genes whose RNAs are homologous to a specific dsRNA in the cell (Bass, 2000). Another targeted response is the specific modification of dsRNA by adenosine deamination. Extended dsRNA duplexes can be hyper-edited by members of the ADAR (adenosine deaminases that act on RNA) family of enzymes (Hough and Bass, 2000). ADARs modify dsRNA by hydrolytic deamination of adenosine (A) to inosine (I), and carry out either selective editing or hyper-editing (Emeson and Singh, 2000; Hough and Bass, 2000). Selective editing, in which a limited number of A to I conversions occur in an mRNA, is a well established mechanism of gene regulation. As inosine residues are recognized by both the translation and splicing machinery as guanosine (G), A to I editing changes the coding potential of mRNAs, and can affect pre-mRNA splicing by introducing new splice sites (Rueter et al., 1999). Hyper-editing occurs on extended RNA duplexes, resulting in up to 50% A to I conversion (Nishikura et al., 1991; Polson and Bass, 1994). Hyper-editing of dsRNAs arising from antisense transcription may be involved in gene regulation, as has been suggested for the Xenopus bFGF gene (Kimelman and Kirschner, 1989; Saccomanno and Bass, 1999) and for polyoma virus (Kumar and Carmichael, 1997). Hyper-editing has also been detected in Drosophila 4f-rnp cDNAs (Petschek et al., 1996), and in hairpin structures in poly(A)+ RNA isolated from Caenorhabditis elegans (Morse and Bass, 1999). Hyper-editing of a number of viral RNAs has also been reported (Emeson and Singh, 2000), including measles virus (Bass et al., 1989; Cattaneo et al., 1989), human parainfluenza virus (Murphy et al., 1991), vesicular stomatitis virus (O'Hara et al., 1984), avian leukosis virus (Hajjar and Linial, 1995), respiratory syncitial virus (Rueda et al., 1994), Rous-associated virus (Felder et al., 1994) and polyoma virus (Kumar and Carmichael, 1997). The cytoplasmic isoform of ADAR1 is inducible by interferon (Patterson and Samuel, 1995), a characteristic of other enzymes that are involved in antiviral defence [e.g. PKR and 2-5A system (RNase L)]. These observations lend weight to the idea that A to I hyper-modification is an antiviral mechanism. Although hyper-editing alone may be efficient in destroying the sense of viral dsRNA, it would still be engaged by the cellular translation machinery and would thus compete with normal cellular protein synthesis. A mechanism to dispose of hyper-edited dsRNAs might enable cells to combat viral infection more effectively. We previously reported a ribonuclease activity in various protein extracts that specifically degraded inosine-containing RNA (I-RNA) (Scadden and Smith, 1997). To study this activity, referred to as I-RNase, we utilized I-RNA substrates where the inosine residues were incorporated by in vitro transcription, rather than by deamination (i.e. involving G to I rather than A to I substitutions). We subsequently found that a number of ribonucleases [e.g. RNase A, S1 nuclease, Rrp41p, Rrp4p (Allmang et al, 1999)] were able to degrade I-RNA much more rapidly than the equivalent guanosine-containing RNA. This suggested that the observed 'I-RNase' activity resulted from destabilization of intramolecular secondary structure within the single-stranded substrate RNAs, making them more generally accessible to a wide variety of ribonucleases. We have now carried out a series of experiments to investigate the fate of deaminated-dsRNA (d-dsRNA) that contains multiple inosine substitutions as a result of in vitro hyper-editing by ADAR2. These RNA substrates were thus essentially equivalent to hyper-edited dsRNA detected in vivo. Upon incubation in various cytoplasmic extracts, d-dsRNA is susceptible to cleavage at specific sites consisting of alternating IU and UI base pairs. In contrast, unmodified dsRNA and even d-dsRNAs that contain four consecutive IU base pairs are not cleaved. Strikingly, dsRNAs in which alternating IU and UI base pairs are replaced by isomorphic GU and UG base pairs (Masquida and Westhof, 2000) are not cleaved. Hyper-editing therefore appears to generate specific RNA structures that might constitute a suitable unique tag for the disposal of viral dsRNA. Results The dsRNA substrate that we initially used to analyse the fate of d-dsRNA was ΔKP, which comprises exons 2 and 3 of the rat α-tropomyosin gene with no intron between (Scadden and Smith, 1997). In each experiment, only one strand of each dsRNA substrate was labelled, and the single-strand RNA (ssRNA) control corresponded to the same strand. The ΔKP dsRNA was deaminated using ADAR2, typically resulting in up to ∼40% A to I conversion (Figure 1A). All substrate RNAs used in this study had a comparable level of deamination (∼40%) unless otherwise stated. Analysis of d-dsRNA by native gel electrophoresis showed that all of the input RNA was deaminated to a similar extent, as indicated by the relatively tight band with lower mobility than unmodified dsRNA (Figure 1B, compare lanes 1 and 8). When ΔKP d-dsRNA was incubated in Xenopus oocyte extract, it was cleaved at a unique position to give two products (Figure 1C, lanes 1–4). In contrast, unmodified ΔKP dsRNA was stable (lanes 5–8), while the equivalent ssRNA was degraded more slowly, yielding numerous degradation products (lanes 9–12). Moreover, the rate of cleavage of d-dsRNA was equal to or often greater than the rate of ssRNA degradation, depending on the individual extract (lanes 1–4, 9–12). The products of cleavage appeared to be stable, which suggested that they remained double stranded. When other d-dsRNA substrates were tested, similar results were obtained, although a more complex pattern of cleavage products was usually observed. Figure 1D shows experiments with polyoma virus (PV) and chloramphenicol acetyl transferase (CAT) RNAs, respectively. When PV d-dsRNA was incubated in the Xenopus extract, it was also less stable than the equivalent dsRNA (compare lanes 2 and 4 of Figure 1D). Similarly, it was degraded more rapidly than the ssRNA equivalent (compare lanes 2 and 6). Although cleavage of PV d-dsRNA yielded more products than ΔKP, they were again stable. Cleavage of the CAT d-dsRNA was more vigorous, giving a complex pattern of cleavage products (lane 8). Nevertheless, the equivalent dsRNA was relatively stable, and the ssRNA was degraded more slowly (compare lanes 8, 10 and 12). Note that in this particular assay a very small amount of specific cleavage of CAT dsRNA was detectable after 90 min (lane 10). This cleavage probably reflects the small amount of Xenopus ADAR activity present in the extract. When a time course of cleavage of CAT RNA was performed, two bands (indicated by asterisks in Figure 1D) were the first cleavage products detected. These then gave rise to other cleavage products (data not shown). Similar data showing specific cleavage were obtained with two other substrates (data not shown). In summary, for all substrates tested, cleavage of the d-dsRNA was enhanced compared with the equivalent dsRNA, yielding a limited number of discrete products. As cleavage of ΔKP was most easily interpreted due to its single cleavage site, this substrate was used as a model to study the specificity of d-dsRNA cleavage. Figure 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 HaeIII, 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). Download figure Download PowerPoint Cleavage by a cytoplasmic ribonuclease Cleavage of d-dsRNA was tested in HeLa cell nuclear (NE) and cytoplasmic (S100) extracts (Figure 2A). Cleavage of ΔKP d-dsRNA occurred in HeLa cell S100 extract, yielding the same major products as in the Xenopus oocyte extract (compare lanes 1–4 with 13–16). In addition, a minor cleavage product was detected (lanes 13–16). No cleavage of dsRNA was detected (lanes 17–20). In contrast, incubation of ΔKP d-dsRNA in HeLa cell NE resulted in negligible cleavage compared with HeLa cell S100 (compare lanes 8 and 16). Equivalent amounts of NE and S100 protein were used and the A260/A280 ratios were identical. Therefore, the difference in activity could not be accounted for by differences in either protein concentration or differential competition by nucleic acids. The minor amount of activity seen in NE (lanes 5–8) can probably be accounted for by cytoplasmic contamination during extract preparation. The ribonuclease activity responsible for specifically cleaving d-dsRNA therefore appears to be cytoplasmic. This is consistent with the previous observation that hyper-edited PV RNA could be detected in the nuclear, but not cytoplasmic, compartment (Kumar and Carmichael, 1997). Figure 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. Download figure Download PowerPoint RNA duplexes containing a GU base pair can be photocleaved in the presence of small organic molecules such as flavin mononucleotide (FMN) or riboflavin (Burgstaller et al., 1997). Since ADAR treatment results in replacement of A–U by IU base pairs, which are isomorphic with GU pairs, it was possible that the d-dsRNAs were being attacked by a similar mechanism. To test this we carried out assays in the dark. Cleavage was identical when carried out in the light or dark (Figure 2B). Moreover, d-dsRNAs were stable in a Xenopus extract that had been heated at 60°C for 10 min prior to incubation or in an assay where the extract was omitted (data not shown). This demonstrates that photocleavage was not responsible for the observed cleavage of d-dsRNAs, but that a ribonuclease was involved. Editing of a specific sequence is required for cleavage We used RT–PCR to amplify the sequences of deaminated ΔKP RNAs. Subsequent sequencing of the cloned RT–PCR products enabled the identification of edited positions in the RNA. The positions of deamination in the sense strand of ΔKP RNA deaminated to 40% are given in bold type in Figure 3A. As cleavage of ΔKP d-dsRNA was limited to one position (yielding two products), it was possible to map the site of cleavage by primer extension using a primer complementary to the 3′ end of the RNA (data not shown). This revealed that cleavage occurred within a sequence that potentially comprised both IU and UI base pairs (boxed; sequence CS below Figure 3A). Subsequent analyses of RT–PCR products corresponding to the antisense strand revealed that the base paired with U in the CS sequence was also inosine, even in d-dsRNAs with only 5% A to I conversion. Thus, cleavage of the d-dsRNA occurred within a sequence comprising four consecutive IU/UI wobble base pairs. Figure 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. Download figure Download PowerPoint Inspection of the sequence in Figure 3A revealed several other positions with adjacent inosines that were not sites for cleavage. In particular, a run of four consecutive AU base pairs (underlined; sequence 4I below Figure 3A) was a hot spot for editing; even at 25% overall levels of deamination, this sequence contained four inosines in half of the sequenced clones and at least three inosines in 90% of clones. If the sole requirement for cleavage was extensive local deamination, one might expect the d-dsRNA to be cleaved at positions with three or four consecutive IU base pairs. This suggested that it was either the presence of alternating IU and UI base pairs that made the RNA susceptible for cleavage, or that the sequence context of the IU base pairs may be important for cleavage. To distinguish between these possibilities, two mutants were generated (Figure 3B). In the mutant ΔU, the U–A base pair in the cleavage site (CS) was mutated to A–U and the downstream site (4I) was unchanged. In the mutant +U, the downstream sequence was mutated to resemble the cleavage site (AAAA to AAUA), while the sequence at the cleavage site was unaltered. The two mutants potentially contained either no cleavage sites (ΔU) or two cleavage sites (+U). The three dsRNAs [wild type (WT), ΔU, +U] were deaminated to the same extent by ADAR2. Consistent with expectations, the ΔU d-dsRNA was not cleaved efficiently in Xenopus oocyte extract (Figure 3C, lanes 1–4), while +U d-dsRNA generated products corresponding to cleavage at both the original site and the mutated downstream site (lanes 7–10). The wild-type ΔKP was cleaved only at the previously identified site (lanes 13 and 14). Primer extension using a labelled primer complementary to the extreme 3′ end of the sense RNA strand was used to confirm the location of the cleavage sites in +U (data not shown). For both mutants no cleavage of dsRNA was observed (lanes 6 and 12). These data demonstrate that cleavage depends on the presence of alternating IU and UI base pairs rather than the position of IU pairs within the RNA. A further series of mutants were generated in which the order of A–U and U–A pairs in the unedited cleavage site were changed (Figure 3D). When each corresponding mutant d-dsRNA was incubated in Xenopus oocyte extract, they were cleaved with varying efficiencies (Figure 3D). Mutants AUAA and AUUA were cleaved even more efficiently than the wild-type AAUA. In contrast, AAAU and UAAA were cleaved to a very small extent. For all mutants, the equivalent dsRNAs were assayed in parallel and they were all stable as expected (data not shown). Although each of the RNAs were deaminated equally by ADAR2 (45% A to I), it is possible that the deamination efficiency of particular positions within the mutated cleavage sites were reduced, thereby reducing the likelihood of cleavage. On the other hand, none of the mutations introduced was likely to dramatically reduce the frequency of deamination within the cleavage site sequence according to either the 5′ or 3′ neighbour preferences of ADAR2 (Polson and Bass, 1994; Lehmann and Bass, 2000). Following the determination of the requirements for cleavage in ΔKP, the sequences of the other test RNAs (Figure 1D) were reanalysed. For the PV RNA, where the cleavage pattern was relatively simple, it was possible to identify the precise sequences of alternating A–U and U–A base pairs that produced the cleavage site after editing. This analysis also revealed that both the A–U content of the RNAs and number of potential cleavage sites increased in the order ΔKP < PV1 < CAT. This order parallels the order of cleavage complexity (see Figure 1). It is possible that some sites are more likely to be deaminated than others according to the neighbour preferences of ADAR2 (Polson and Bass, 1994; Lehmann and Bass, 2000) and would therefore constitute better targets for cleavage. The precise cleavage site in d-dsRNA was mapped using 20 bp synthetic RNA duplexes that contained alternating IU base pairs corresponding to the cleavage site in ΔKP (Figure 4A and B; see Materials and methods). In each experiment, only one of the two strands was 5′ end-labelled. Complete hybridization of the labelled strand was confirmed by native gel electrophoresis (data not shown). Cleavage of the sense strand occurred 5′ of the U in the sequence 5′-IpU-3′, while the other strand was cleaved in five positions—predominantly 5′ of the two U residues opposite the two consecutive inosines (Figure 4B). The cleavage products appear to have 3′ phosphate ends since they co-migrated with the alkaline hydrolysis products (Figure 4A, lanes 3 and 6). This was confirmed by periodate oxidation and beta-elimination analysis. The full-length RNA was shortened by one nucleotide after beta-elimination, while the cleavage products were unaffected, showing that they do not possess a 3′ hydroxyl group (data not shown). Figure 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. Download figure Download PowerPoint Sequences containing GU and UG base pairs are not cleaved Inosine differs from guanosine only by the absence of the N2 exocyclic amine group (circled in Figure 4E). IU and UI wobble base pairs are therefore isomorphic with GU and UG wobble base pairs, respectively, although they are slightly less stable (Strobel et al., 1994). Due to the asymmetry of the glycosidic bond angle between the base and C1′ sugar atom in a GU pair compared with a Watson–Crick base pair, GU and UG pairs are not isomorphic with each other. In addition, the presence of tandem GU or UG pairs in a duplex causes overwinding or underwinding of the RNA helix depending on the order of the wobble base pairs. This causes large and distinctive conformational changes in the RNA duplex (Masquida and Westhof, 2000; Varani and McClain, 2000). It is likely that consecutive IU and UI base pairs would cause very similar distortions in the dsRNA structure. Such structural distortions could be responsible for the specific cleavage of d-dsRNA at alternating IU/UI base pairs, but not at runs of IU base pairs (Figure 3). To test whether the cleavage of d-dsRNA is related only to the distortion of the dsRNA geometry, we made ΔKP dsRNA in which IU and UI were replaced by GU and UG base pairs (GU-dsRNA). The GU-dsRNAs were prepared by in vitro transcription using templates derived by RT–PCR from d-dsRNA (see Materials and methods). The d-dsRNA and GU-dsRNA were then assayed for cleavage in Xenopus oocyte extract (Figure 4C). While d-dsRNA was cleaved (lanes 1–4), the GU-dsRNA was stable (lanes 5–8). This observation is made even more significant by the fact that only a proportion of the d-dsRNA has the correct sequence necessary for cleavage (by deamination), while 100% of the GU-dsRNAs contain GU and UG base pairs at the cleavage site. Therefore, cleavage of the d-dsRNA cannot be replicated in dsRNAs containing equivalent GU distortions. To analyse this further we compared cleavage of short synthetic RNA duplexes that contained either IU and UI base pairs or GU and UG base pairs (Figure 4D). The sequence of the short dsRNAs (20 bp duplex) comprised the cleavage site in ΔKP plus flanking nucleotides (see Materials and methods). When these RNAs were incubated in Xenopus oocyte extract, the IU duplex was cleaved while the GU-dsRNA was stable (Figure 4D; compare lanes 1–4 and 5–8). Moreover, both strands of the inosine-containing dsRNA were cleaved while neither strand of the guanosine-containing dsRNA was cut (data not shown). The cleavage of the short synthetic RNAs not only confirmed the results obtained with long RNA duplexes, but also demonstrated that inosine is required only at the cleavage site. There are two obvious explanations for the resistance of the GU substrate to cleavage. First, the GU and IU duplexes may have an identical overall structure, but the additional minor groove amine groups on guanosine might interfere with cleavage. Alternatively, the IU duplex may adopt a different structure from the GU duplex, which at the extreme could be locally melted around the IU base pairs, giving rise to the differential cleavage. To address these possibilities, the stability of the 20 bp synthetic duplexes was analysed by RNA melting experiments (Loakes and Brown, 1994). Both duplexes showed a single A260 transition with melting temperatures of 63.3 and 60.0°C for the GU and IU duplexes, respectively (assays in 10 mM HEPES pH 7.5, 100 mM NaCl). This is consistent with previous reports that IU base pairs are less stable than GU (Strobel et al., 1994). The modest difference in melting temperature does not appear sufficient to explain the complete lack of cleavage of GU RNA at 25°C while IU RNA is efficiently cleaved. Thus, cleavage of hyper-edited RNA in cytoplasmic extracts appears to involve recognition of specific distorted dsRNA structures. Discussion The occurrence of extended dsRNA in cells is relatively uncommon, and is frequently associated with infection by DNA or RNA viruses. Antiviral defence mechanisms such as the PKR and 2′-5′-adenylate synthase/RNase L pathways are stimulated by the presence of dsRNA. An additional mechanism to remove dsRNA from cells may involve hyper-editing of dsRNA by ADARs, followed by targeted cleavage. The data presented here clearly demonstrate that various cell extracts contain an endoribonuclease activity that fulfils the requirements for the second step in this proposed pathway. Co