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RNAi is antagonized by A→I hyper‐editing

RNAi is antagonized by A→I hyper‐editing EMBO reports A.D.J. Scadden & Christopher W.J. Smith Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK Received August 31, 2001; revised October 1, 2001; accepted October 9, 2001 RNA interference (RNAi) and adenosine to inosine conversion by mobile genetic elements such as viruses, transposable are both mechanisms that respond to double-stranded RNA elements or transgenes. Degradation of the target mRNA is (dsRNA) and have been suggested to have antiviral roles. restricted to the region that corresponds to the dsRNA (Zamore RNAi involves processing of dsRNA to short interfering RNAs et al., 2000). Initially, the dsRNA is processed to generate short (siRNAs), which subsequently mediate degradation of the RNA fragments of 21–23 nt, referred to as small interfering RNAs cognate mRNAs. Deamination of adenosines changes the (siRNAs) (Zamore et al., 2000; Elbashir et al., 2001). The highly coding capacity of the RNA, as inosine is decoded as guano- conserved class of RNase III homologues, represented by the sine, and alters the structure because A–U base pairs are Drosophila enzyme Dicer, have been implicated in processing replaced by I•U wobble pairs. Here we show that RNAi is of dsRNA to siRNAs (Bernstein et al., 2001; Grishok et al., 2001; inhibited if the triggering dsRNA is first deaminated by ADAR2. Knight and Bass, 2001). Subsequently, the siRNAs are thought to Moreover, we show that production of siRNAs is progressively act as guide sequences within a multicomponent nuclease for inhibited with increasing deamination and that this is sufficient to targeted degradation of the mRNA (Hammond et al., 2001). explain the inhibition of RNAi upon hyper-editing of dsRNAs. dsRNAs are also targets for hyper-editing by ADARs (Emeson and Singh, 2000; Hough and Bass, 2000). ADARs catalyse the hydrolytic deamination of adenosine (A) to inosine (I). This has INTRODUCTION two important consequences. First, the coding capacity of the Extended double-stranded RNA (dsRNA) duplexes are unusual RNA is changed, since I is recognized as G during translation. in eukaryotic cells and are often indicative of viral infection or Secondly, the structure of the dsRNA is altered by the replace- the presence of other mobile genetic elements. A number of ment of Watson–Crick A–U base pairs by I•U wobble pairs, mechanisms exist to respond to dsRNA. For example, the PKR which are isomorphic with G•U base pairs (Masquida and and RNase L/oligo 2-5 A synthase systems act to globally inhibit Westhof, 2000). The change in coding capacity is important gene expression in vertebrate cells (Silverman et al., 1997; during specific editing of mRNAs, in which a limited number of Samuel, 1998). In contrast, RNA interference (RNAi) and adeno- A→I changes, guided by short intramolecular base-paired sine deamination by ADARs (adenosine deaminases that act on segments, alter specific amino acids within the encoded protein. In dsRNA) directly target the dsRNA with covalent processing reac- contrast, hyper-editing of long dsRNA results in up to 50% of tions and coexist throughout the metazoa (Bass, 2000; Hough adenosine residues being replaced by inosine, resulting in multiple and Bass, 2000; Carthew, 2001; Sharp, 2001). mis-sense alterations. Moreover, the specific structures induced by the wobble I•U base pairs or the presence of inosine per se may RNAi is the post-transcriptional gene silencing (PTGS) mecha- also tag the dsRNA for specific degradation (Scadden and Smith, nism whereby dsRNA directs the specific degradation of cognate mRNA (see Bass, 2000; Carthew, 2001; Sharp, 2001 for 2001) or nuclear retention (Zhang and Carmichael, 2001). reviews). Following the discovery of RNAi in Caenorhabditis Although both RNAi and ADARs are thought to have antiviral elegans (Fire et al., 1998), it was subsequently observed in a roles, it is likely that the two processes are mutually antagonistic. variety of eukaryotic organisms. Moreover, parallels have been Conversion of dsRNA to siRNA would antagonize the activity of drawn between RNAi and the PTGS mechanisms of co-suppres- ADARs, because siRNA duplexes are expected to be too short sion in plants and quelling in fungi. PTGS in plants has been for adenosine deamination (Hough and Bass, 2000). Likewise, associated with antiviral defence (Hamilton and Baulcombe, hyper-editing could antagonize RNAi at two levels (Bass, 2000). 1999). It is likely that RNAi exists to counteract invasion of cells First, hyper-editing by ADARs is likely to alter the structure of the Corresponding author. Tel: +44 1223 333655; Fax: +44 1223 766002; E-mail: [email protected] © 2001 European Molecular Biology Organization EMBO reports vol. 2 | no. 12 | pp 1107–1111 | 2001 1107 scientific reports A.D.J. Scadden & C.W.J. Smith dsRNA sufficiently that it is a poor substrate for siRNA produc- tion. Secondly, the mRNA targeting step of RNAi might be inhib- ited by the substitution of I•U for A–U base pairs between the siRNAs and the target mRNA. Finally, I•U base pairs within the siRNA duplexes themselves might be inhibitory for RNAi. We have now tested the proposed antagonism between ADARs and RNAi. We show that hyper-editing by ADAR2 antagonizes RNAi in vitro and that the inhibition of RNAi is accompanied by a significant decrease in the production of siRNAs from deami- nated dsRNA. This suggests that resistance to RNAi in some tissues (e.g. neuronal cells in C. elegans) could be explained in part by high ADAR activity. RESULTS AND DISCUSSION RNAi is antagonized by A→I editing We investigated RNAi in vitro using the Drosophila extract described previously (Tuschl et al., 1999; Zamore et al., 2000). The target mRNA comprised ∼850 nt of the bacterial chloram- phenicol acetyl transferase (CAT) gene at a concentration of 50 pM. The homologous dsRNA used to trigger RNAi was 595 Fig. 1. RNAi in Drosophila extract. (A) Unlabelled RNAs (dsRNA, d-dsRNA bp in length beginning at the 5′ end of the target. A deaminated- and ssRNA) were assayed for their ability to activate RNAi. These RNAs (10 nM) were preincubated in Drosophila extract for 15 min at 25°C before dsRNA (d-dsRNA) trigger was prepared by hyper-editing in vitro adding 50 pM of each of the target and control RNAs (CAT and ΔKP, with recombinant ADAR2 to give ∼50% A→I conversion. respectively). The assay was continued for a further 15, 30 or 45 min and the Finally, a control trigger single-stranded RNA (ssRNA) corres- products analysed on a 5% polyacrylamide gel. While dsRNA was able to activate RNAi (lanes 1–3), neither d-dsRNA or ssRNA were active. (B) The ponded to the sense strand of the CAT target. Typically, the data from (A) is expressed as a ratio of CAT/ΔKP [+dsRNA (squares) or trigger RNAs were uniformly labelled at a very low specific +d-dsRNA (circles)] relative to the ratio of CAT/ΔKP +ssRNA. The graphical activity and were added to 10 nM. While the CAT target RNA representation of these data reinforces the observation that d-dsRNA is unable was degraded at a similar rate to a control ssRNA (ΔKP) in the to activate RNAi. (C) dsRNA, d-dsRNA and ssRNA (10 nM) were again teste presence of single-stranded CAT trigger RNA (Figure 1A, lanes for their ability to activate RNAi. Three different control RNAs (VLE, ΔKP and PV) and the target RNA (CAT) were used (50 pM each RNA). Time 9–12), in the presence of dsRNA trigger the CAT RNA, but not points were taken at 0, 30 and 60 minutes. Again, only dsRNA was able to the control, was degraded at an enhanced rate [lanes 1–4, activate RNAi (lanes 1–3). Note that the ∼600 nt band in this panel represents and illustrated graphically in Figure 1B (squares)]. In contrast, the trigger RNA, which was labelled to higher specific activity than in the other the d-dsRNA trigger (∼50% deamination) was completely experiments. (D) The data from (C) were expressed as a ratio of CAT/control ((+dsRNA or d-dsRNA) for each control RNA) relative to the ratio of CAT/ inactive in RNAi [Figure 1A, lanes 5–8, Figure 1B (circles)]. control (+ssRNA) for each control RNA: CAT/VLE (triangles), CAT/ΔKP Even when the time-course was extended to 180 min, no (squares), CAT/PV (circles). Data-points for dsRNA are joined by solid lines, significant RNAi activity was observed with d-dsRNA (data not and for d-dsRNA by dashed lines. Approximately the same RNAi activity was shown). To ensure the specificity of the in vitro RNAi, the assay observed regardless of the control RNA used to calculate the CAT/control ratio and the d-dsRNA was again ineffective at activating RNAi. was repeated using three different control RNAs: VLE (445 nt), ΔKP (300 nt) and PV1 (230 nt) (Figure 1C and D). The degradation of the target RNA (with either dsRNA or d-dsRNA) was quantitated relative to each of the control RNAs and normalized to the ratio of target/control degradation observed in the presence of 10 nM ssRNA (Figure 1D). These data tight bands with lower mobility than unmodified dsRNA confirm that, while dsRNA is potent in inducing RNAi, d-dsRNA is (Figure 2A). An RNAi assay was carried out for 90 min where 10 inactive. nM CAT dsRNA, ssRNA or each d-dsRNA was used to activate In the experiments shown in Figure 1, the d-dsRNA was maxi- RNAi against the CAT target in the presence of an unrelated mally edited, with ∼50% of A residues converted to I. RNAs control RNA (ΔKP) (Figure 2B). The data were quantitated and edited more moderately would have a less disrupted structure are represented graphically in Figure 2C, as the ratio of and may be partially active in RNAi. We therefore tested the remaining CAT/ΔKP RNAs normalized to the CAT/ΔKP ratio in ability of d-dsRNAs edited to various levels to activate RNAi and the presence of ssRNA. Again dsRNA but not ssRNA trigger to generate siRNAs (Figure 2). The amount of A→I conversion caused a decrease in the amount of CAT target RNA (compare was quantitated by phosphorImaging following digestion with lanes 1 and 7). As the amount of editing of the d-dsRNA RNase P1 and TLC (data not shown). The d-dsRNAs subse- increased, a corresponding decrease in its effectiveness to quently used in the RNAi assays contained 5, 12, 19, 25 and induce RNAi was seen (lanes 2–6). Thus, increasing levels of 43% A→I conversion. The d-dsRNAs were analysed by native deamination progressively impair the ability of d-dsRNA to gel electrophoresis to verify that all of the input RNA was deaminated to a similar extent, as indicated by the relatively induce RNAi. 1108 EMBO reports vol. 2 | no. 12 | 2001 scientific reports [α- P]ATP and incubated them under identical conditions as used in the RNAi assay shown in Figure 2B. Incubation of dsRNA in the Drosophila extract gave rise to siRNAs, while no siRNAs were generated from ssRNA (Figure 2D, compare lanes 1 and 7). In this experiment ∼5% of the input dsRNA was proc- essed to siRNAs, while in other experiments up to 15% conver- sion of input dsRNA was observed (data not shown). A progressive decrease in siRNA production was observed to accompany the increase in editing of the d-dsRNA (lanes 2–6), with only background levels detectable at 43% deamination. The reduced levels of siRNA are sufficient to account for the abolition of RNAi at high levels of deamination. siRNAs contain a similar amount of inosine as input RNAs It is possible, at intermediate levels of editing (e.g. 19 % A→I conversion), where siRNA production is reduced only ∼5-fold, that later steps in RNAi may also be affected if the siRNAs include A→I modifications. However, it is possible that the Dicer ribonuclease involved in processing the dsRNA may discriminate against sequences containing I residues due to the altered structure. We therefore carried out an analysis to deter- mine the inosine content of siRNAs generated from d-dsRNA that contained 18% A→I conversion. dsRNA and d-dsRNA were incubated in Drosophila extract for 90 min at 25°C to allow production of siRNAs. The siRNAs and unprocessed RNA from both dsRNA and d-dsRNA were subsequently recovered following denaturing gel electrophoresis. The recovered RNAs were digested with RNase P1 and then subjected to TLC to sepa- Fig. 2. A→I editing progressively inhibits RNAi and production of siRNAs. rate 5′-IMP from 5′-AMP (Figure 3). Prior to incubation in (A) CAT dsRNA was edited to varying degrees (5, 12, 19, 25 and 43% A→I) Drosophila extract, 18% of the A residues in the input d-dsRNA using ADAR2 and analysed by native gel electrophoresis. As the level of were converted to I, while the dsRNA contained no I (lanes 5 editing increased there was a corresponding reduction in their mobility on a native gel (for example, compare lanes 1 and 6). Each deaminated RNA was and 6). The siRNAs produced from d-dsRNA contained 15% A→I relatively homogeneous, as indicated by the relatively tight bands with lower conversion (lane 1), only marginally less than the 18% conver- mobility than unmodified dsRNA. (B) An RNAi assay was performed for sion in the full-length d-dsRNA recovered from the same lane of 90 min where each CAT d-dsRNA was tested for its ability to activate RNAi the denaturing gel (lane 2), and the input d-dsRNA (lane 5). No (lanes 2–6) alongside dsRNA (lane 1) and ssRNA (lane 7). In this assay the control RNA used was ΔKP. As the level of editing increased in the inosine was detectable (<0.8%) in the siRNAs generated from d-dsRNAs, there was a clear reduction in the amount of RNAi (compare lanes the dsRNA or unprocessed dsRNA (lanes 3 and 4). In contrast, 1 and 6). (C) The data from (B) are represented as the CAT/ΔKP ratio relative Zamore et al. (2000) observed ∼3–6% A→I conversion of the ΔKP ratio in the presence of ssRNA. In order to accurately to the CAT/ dsRNA during their RNAi experiments, but <0.7% in the siRNAs. quantitate the amount of target RNA remaining in the presence of highly edited d-dsRNA (lanes 4–6), it was necessary to determine the sum of the two upper This is consistent with our observations if a small proportion of bands that correspond to CAT RNA (marked with an asterisk in lane 6). The their dsRNA were heavily edited and thus unable to generate anomalous mobility of a small proportion of the CAT target RNA is the result siRNAs. The reason for the lower level of endogenous ADAR of partial hybridization of the CAT target RNA with the 200-fold excess of activity in our experiments is not clear. It could be related to the unlabelled CAT d-dsRNA. An inverse correlation exists between the level of editing in d-dsRNA and its ability to induce RNAi. (D) The production of age of the embryos used to prepare the extract (3–6 h as opposed siRNAs from CAT dsRNA and d-dsRNAs was analysed using RNAs labelled to 0–2 h) or to slight differences in the quantity of extract or dura- with [α P]ATP. This assay was performed alongside that shown in (B) using tion of the RNAi assay. Our data show that moderately deami- identical conditions. As the level of editing increased, the production of nated dsRNA can be processed to siRNAs with no strong siRNAs showed a corresponding decrease (compare lanes 1 and 6). The percentage of the input RNA that was converted to siRNAs is shown below discrimination against the generation of siRNAs containing I•U each lane. base pairs. A 15% level of A→I conversion corresponds to an average of 0.8 inosines per siRNA, a level at which ∼55% of the siRNAs would be expected to contain at least one inosine. If perfect Watson–Crick base-pairing between siRNAs and the targeted RNAs is required, the presence of I in the siRNAs will impair RNAi disproportionately more than can be accounted for Editing of RNA reduces the production of siRNAs by the reduced amounts of siRNA. This could be tested directly To test whether hyper-editing directly inhibits the production of by carrying out in vitro RNAi experiments with synthetic siRNAs siRNAs, we labelled trigger RNAs to high specific activity with containing single A–U to I•U substitutions. EMBO reports vol. 2 | no. 12 | 2001 1109 scientific reports A.D.J. Scadden & C.W.J. Smith localization appears likely (Fire et al., 1998). Thus, in some circumstances the two systems may be able to compete directly for access to dsRNA, while elsewhere competition might be determined by which system is first to encounter the dsRNA and thus convert it to a form that cannot be recognized by the other system. METHODS RNAs. All RNAs were prepared by in vitro transcription. The CAT target RNA and the control RNAs were labelled inter- nally with [α- P]UTP (3000 Ci/mmol; Amersham), where Fig. 3. siRNAs contain inosine. dsRNA and d-dsRNA (with 18% A→I) were 50 μCi [α- P]UTP was added per 50 μl transcription incubated in Drosophila extract for 90 min at 25°C. The siRNAs (and reaction. The ‘unlabelled’ trigger RNAs were trace-labelled unprocessed RNA) generated from both dsRNA and d-dsRNA were recovered with [α- P]UTP (3000 Ci/mmol; Amersham) at a specific following gel electrophoresis and analysed for inosine content by RNase P1 activity at least 250-fold lower than that of the target RNA. The digestion followed by TLC. The input d-dsRNA contained 18% A→I conversion (lane 5), while the dsRNA contained no inosine (lane 6). labelled trigger RNAs were labelled internally (on the sense Following recovery from the gel, the unprocessed dsRNA (L) and siRNA strand only) with [α- P]ATP (3000 Ci/mmol; Amersham) to a generated from dsRNA contained no inosine (lanes 3 and 4). In contrast, similar specific activity as the target RNAs. All of the RNAs were unprocessed d-dsRNA (L) contained an equivalent amount of inosine as in initiated using an m G(5′)ppp(5′)G dinucleotide primer. dsRNAs the input d-dsRNA (18% A→I, lane 2). SiRNAs generated from d-dsRNA contained inosine at a level that corresponds to 15% A→I conversion. were prepared by annealing complementary RNAs (Scadden and Smith, 2001). ΔKP RNA (Scadden and Smith, 1997) was synthesized using SP6 RNA polymerase following linearization with BamHI to give a 296-nt transcript. PV 1 RNA (Scadden and Smith, 2001) was Our experiments confirm the predicted biochemical antago- synthesized using T7 RNA polymerase following linearization nism between ADARs and RNAi (Bass, 2000). We used human with BamHI to give a 230-nt transcript. CAT target RNA ADAR2, but similar results would be expected with other (O’Connell and Keller, 1994) was synthesized using T7 RNA poly- ADARs, which show similar maximal levels of ∼50% A→I merase after linearizing the plasmid with PvuII to give an 858-nt conversion upon hyper-editing of long dsRNA substrates (Hough transcript. CAT dsRNA was prepared as described previously and Bass, 2000). Even if there were some differences in the pref- (Scadden and Smith, 2001). VLE RNA comprised nucleotides erences for editing particular adenosines, the overall structural 1383–1813 from the Vg1 cDNA (Pressman-Schwartz et al., 1992). distortions in the dsRNA would be similar, resulting in lack of RNAi assay. The RNAi assay was performed using the conditions processing by Dicer enzymes. At present, it is not clear what described previously (Tuschl et al., 1999; Zamore et al., 2000). physiological consequences result from this antagonism. Expres- The Drosophila extract was prepared from 3–6 h PEL embryos sion of ADARs in various organisms is highest in neural tissues according to the method of Becker et al. (1994). Electrophoresis and also in the developing vulva of C. elegans (L. Tonkin and B. on a 15% polyacrylamide gel enabled the analysis of siRNAs. Bass, personal communication). RNAi in most C. elegans tissues Data were quantitated following phosphorImaging using can be readily achieved by soaking the worms in dsRNA or feeding Imagequant software (Molecular Dynamics). with Escherichia coli strains that express a trigger dsRNA. In Deamination reaction. Deamination reactions were carried out contrast, neurons are relatively resistant (Kamath et al., 2000; as described previously using recombinant human ADAR2 Maeda et al., 2001), and efficient RNAi of neuronally expressed (Scadden and Smith, 2001). The time of the deamination genes has only been achieved by in vivo expression of a herit- reaction was adjusted to achieve varying levels of editing. able inverted repeat gene corresponding to the target gene under Deamination reactions using labelled and unlabelled dsRNAs the control of a strong heat shock-inducible promoter were carried out in parallel to enable quantitation of unlabelled (Tavernarakis et al., 2000). Likewise, injection of dsRNA was d-dsRNAs. To analyse the efficiency of the deamination unable to invoke RNAi in the vulva (Fire et al., 1998). It is reactions, 50 fmol of RNA were digested with RNase P1 and tempting to speculate that the high levels of ADARs in neurons analysed by TLC (Scadden and Smith, 2001). and vulva can explain, at least in part, the relative resistance of genes in these tissues to RNAi (Bass, 2000). This should be ACKNOWLEDGEMENTS readily testable with ADAR knockout strains of C. elegans. Both RNAi and ADARs have been suggested to have roles in We would like to thank Dr Karl Peter Nightingale, Walter Keller antiviral defence, although both systems also have roles in and Anna Git for gifts of reagents, and Brenda Bass for sharing normal development (Emeson and Singh, 2000; Hough and unpublished data and for comments on the manuscript. We Bass, 2000; Grishok et al., 2001; Hutvágner et al., 2001). would also like to thank Mary O’Connell and Witek Filipowicz Mammalian ADARs are usually confined to the nucleus, for comments on the manuscript. This work was supported by a although an interferon inducible ADAR1 isoform is cytoplasmic grant from the Wellcome Trust (052241). A.D.J.S. was also (Patterson and Samuel, 1995). The subcellular localization of the supported by a Research Fellowship from Newnham College, RNAi machinery has not been investigated, but cytoplasmic University of Cambridge. 1110 EMBO reports vol. 2 | no. 12 | 2001 scientific reports Maeda, I., Kohara, Y., Yamamoto, M. and Sugimoto, A. (2001) Large-scale REFERENCES analysis of gene function in Caenorhabditis elegans by high-throughput Bass, B.L. (2000) Double-stranded RNA as a template for gene silencing. RNAi. Curr. Biol., 11, 171–176. Cell, 101, 235–238. Masquida, B. and Westhof, E. (2000) On the wobble G•U and related pairs. Becker, P.B., Tsukiyama, T. and Wu, C. (1994) Chromatin assembly extracts RNA, 6, 9–15. O’Connell, M.A. and Keller, W. (1994) Purification and properties of double- from Drosophila embryos. 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(2001) A role for the RNase III enzyme DCR-1 promiscuously A-to-I edited RNAs. Cell, 106, 465–475. in RNA interference and germ line development in C. elegans. Science, 293, 2269–2271. DOI: 10.1093/embo-reports/kve244 EMBO reports vol. 2 | no. 12 | 2001 1111 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png EMBO Reports Springer Journals

RNAi is antagonized by A→I hyper‐editing

Scadden, A D J; Smith, Christopher W J
EMBO Reports , Volume 2 (12) – Dec 1, 2001
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Abstract

EMBO reports A.D.J. Scadden & Christopher W.J. Smith Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK Received August 31, 2001; revised October 1, 2001; accepted October 9, 2001 RNA interference (RNAi) and adenosine to inosine conversion by mobile genetic elements such as viruses, transposable are both mechanisms that respond to double-stranded RNA elements or transgenes. Degradation of the target mRNA is (dsRNA) and have been suggested to have antiviral roles. restricted to the region that corresponds to the dsRNA (Zamore RNAi involves processing of dsRNA to short interfering RNAs et al., 2000). Initially, the dsRNA is processed to generate short (siRNAs), which subsequently mediate degradation of the RNA fragments of 21–23 nt, referred to as small interfering RNAs cognate mRNAs. Deamination of adenosines changes the (siRNAs) (Zamore et al., 2000; Elbashir et al., 2001). The highly coding capacity of the RNA, as inosine is decoded as guano- conserved class of RNase III homologues, represented by the sine, and alters the structure because A–U base pairs are Drosophila enzyme Dicer, have been implicated in processing replaced by I•U wobble pairs. Here we show that RNAi is of dsRNA to siRNAs (Bernstein et al., 2001; Grishok et al., 2001; inhibited if the triggering dsRNA is first deaminated by ADAR2. Knight and Bass, 2001). Subsequently, the siRNAs are thought to Moreover, we show that production of siRNAs is progressively act as guide sequences within a multicomponent nuclease for inhibited with increasing deamination and that this is sufficient to targeted degradation of the mRNA (Hammond et al., 2001). explain the inhibition of RNAi upon hyper-editing of dsRNAs. dsRNAs are also targets for hyper-editing by ADARs (Emeson and Singh, 2000; Hough and Bass, 2000). ADARs catalyse the hydrolytic deamination of adenosine (A) to inosine (I). This has INTRODUCTION two important consequences. First, the coding capacity of the Extended double-stranded RNA (dsRNA) duplexes are unusual RNA is changed, since I is recognized as G during translation. in eukaryotic cells and are often indicative of viral infection or Secondly, the structure of the dsRNA is altered by the replace- the presence of other mobile genetic elements. A number of ment of Watson–Crick A–U base pairs by I•U wobble pairs, mechanisms exist to respond to dsRNA. For example, the PKR which are isomorphic with G•U base pairs (Masquida and and RNase L/oligo 2-5 A synthase systems act to globally inhibit Westhof, 2000). The change in coding capacity is important gene expression in vertebrate cells (Silverman et al., 1997; during specific editing of mRNAs, in which a limited number of Samuel, 1998). In contrast, RNA interference (RNAi) and adeno- A→I changes, guided by short intramolecular base-paired sine deamination by ADARs (adenosine deaminases that act on segments, alter specific amino acids within the encoded protein. In dsRNA) directly target the dsRNA with covalent processing reac- contrast, hyper-editing of long dsRNA results in up to 50% of tions and coexist throughout the metazoa (Bass, 2000; Hough adenosine residues being replaced by inosine, resulting in multiple and Bass, 2000; Carthew, 2001; Sharp, 2001). mis-sense alterations. Moreover, the specific structures induced by the wobble I•U base pairs or the presence of inosine per se may RNAi is the post-transcriptional gene silencing (PTGS) mecha- also tag the dsRNA for specific degradation (Scadden and Smith, nism whereby dsRNA directs the specific degradation of cognate mRNA (see Bass, 2000; Carthew, 2001; Sharp, 2001 for 2001) or nuclear retention (Zhang and Carmichael, 2001). reviews). Following the discovery of RNAi in Caenorhabditis Although both RNAi and ADARs are thought to have antiviral elegans (Fire et al., 1998), it was subsequently observed in a roles, it is likely that the two processes are mutually antagonistic. variety of eukaryotic organisms. Moreover, parallels have been Conversion of dsRNA to siRNA would antagonize the activity of drawn between RNAi and the PTGS mechanisms of co-suppres- ADARs, because siRNA duplexes are expected to be too short sion in plants and quelling in fungi. PTGS in plants has been for adenosine deamination (Hough and Bass, 2000). Likewise, associated with antiviral defence (Hamilton and Baulcombe, hyper-editing could antagonize RNAi at two levels (Bass, 2000). 1999). It is likely that RNAi exists to counteract invasion of cells First, hyper-editing by ADARs is likely to alter the structure of the Corresponding author. Tel: +44 1223 333655; Fax: +44 1223 766002; E-mail: [email protected] © 2001 European Molecular Biology Organization EMBO reports vol. 2 | no. 12 | pp 1107–1111 | 2001 1107 scientific reports A.D.J. Scadden & C.W.J. Smith dsRNA sufficiently that it is a poor substrate for siRNA produc- tion. Secondly, the mRNA targeting step of RNAi might be inhib- ited by the substitution of I•U for A–U base pairs between the siRNAs and the target mRNA. Finally, I•U base pairs within the siRNA duplexes themselves might be inhibitory for RNAi. We have now tested the proposed antagonism between ADARs and RNAi. We show that hyper-editing by ADAR2 antagonizes RNAi in vitro and that the inhibition of RNAi is accompanied by a significant decrease in the production of siRNAs from deami- nated dsRNA. This suggests that resistance to RNAi in some tissues (e.g. neuronal cells in C. elegans) could be explained in part by high ADAR activity. RESULTS AND DISCUSSION RNAi is antagonized by A→I editing We investigated RNAi in vitro using the Drosophila extract described previously (Tuschl et al., 1999; Zamore et al., 2000). The target mRNA comprised ∼850 nt of the bacterial chloram- phenicol acetyl transferase (CAT) gene at a concentration of 50 pM. The homologous dsRNA used to trigger RNAi was 595 Fig. 1. RNAi in Drosophila extract. (A) Unlabelled RNAs (dsRNA, d-dsRNA bp in length beginning at the 5′ end of the target. A deaminated- and ssRNA) were assayed for their ability to activate RNAi. These RNAs (10 nM) were preincubated in Drosophila extract for 15 min at 25°C before dsRNA (d-dsRNA) trigger was prepared by hyper-editing in vitro adding 50 pM of each of the target and control RNAs (CAT and ΔKP, with recombinant ADAR2 to give ∼50% A→I conversion. respectively). The assay was continued for a further 15, 30 or 45 min and the Finally, a control trigger single-stranded RNA (ssRNA) corres- products analysed on a 5% polyacrylamide gel. While dsRNA was able to activate RNAi (lanes 1–3), neither d-dsRNA or ssRNA were active. (B) The ponded to the sense strand of the CAT target. Typically, the data from (A) is expressed as a ratio of CAT/ΔKP [+dsRNA (squares) or trigger RNAs were uniformly labelled at a very low specific +d-dsRNA (circles)] relative to the ratio of CAT/ΔKP +ssRNA. The graphical activity and were added to 10 nM. While the CAT target RNA representation of these data reinforces the observation that d-dsRNA is unable was degraded at a similar rate to a control ssRNA (ΔKP) in the to activate RNAi. (C) dsRNA, d-dsRNA and ssRNA (10 nM) were again teste presence of single-stranded CAT trigger RNA (Figure 1A, lanes for their ability to activate RNAi. Three different control RNAs (VLE, ΔKP and PV) and the target RNA (CAT) were used (50 pM each RNA). Time 9–12), in the presence of dsRNA trigger the CAT RNA, but not points were taken at 0, 30 and 60 minutes. Again, only dsRNA was able to the control, was degraded at an enhanced rate [lanes 1–4, activate RNAi (lanes 1–3). Note that the ∼600 nt band in this panel represents and illustrated graphically in Figure 1B (squares)]. In contrast, the trigger RNA, which was labelled to higher specific activity than in the other the d-dsRNA trigger (∼50% deamination) was completely experiments. (D) The data from (C) were expressed as a ratio of CAT/control ((+dsRNA or d-dsRNA) for each control RNA) relative to the ratio of CAT/ inactive in RNAi [Figure 1A, lanes 5–8, Figure 1B (circles)]. control (+ssRNA) for each control RNA: CAT/VLE (triangles), CAT/ΔKP Even when the time-course was extended to 180 min, no (squares), CAT/PV (circles). Data-points for dsRNA are joined by solid lines, significant RNAi activity was observed with d-dsRNA (data not and for d-dsRNA by dashed lines. Approximately the same RNAi activity was shown). To ensure the specificity of the in vitro RNAi, the assay observed regardless of the control RNA used to calculate the CAT/control ratio and the d-dsRNA was again ineffective at activating RNAi. was repeated using three different control RNAs: VLE (445 nt), ΔKP (300 nt) and PV1 (230 nt) (Figure 1C and D). The degradation of the target RNA (with either dsRNA or d-dsRNA) was quantitated relative to each of the control RNAs and normalized to the ratio of target/control degradation observed in the presence of 10 nM ssRNA (Figure 1D). These data tight bands with lower mobility than unmodified dsRNA confirm that, while dsRNA is potent in inducing RNAi, d-dsRNA is (Figure 2A). An RNAi assay was carried out for 90 min where 10 inactive. nM CAT dsRNA, ssRNA or each d-dsRNA was used to activate In the experiments shown in Figure 1, the d-dsRNA was maxi- RNAi against the CAT target in the presence of an unrelated mally edited, with ∼50% of A residues converted to I. RNAs control RNA (ΔKP) (Figure 2B). The data were quantitated and edited more moderately would have a less disrupted structure are represented graphically in Figure 2C, as the ratio of and may be partially active in RNAi. We therefore tested the remaining CAT/ΔKP RNAs normalized to the CAT/ΔKP ratio in ability of d-dsRNAs edited to various levels to activate RNAi and the presence of ssRNA. Again dsRNA but not ssRNA trigger to generate siRNAs (Figure 2). The amount of A→I conversion caused a decrease in the amount of CAT target RNA (compare was quantitated by phosphorImaging following digestion with lanes 1 and 7). As the amount of editing of the d-dsRNA RNase P1 and TLC (data not shown). The d-dsRNAs subse- increased, a corresponding decrease in its effectiveness to quently used in the RNAi assays contained 5, 12, 19, 25 and induce RNAi was seen (lanes 2–6). Thus, increasing levels of 43% A→I conversion. The d-dsRNAs were analysed by native deamination progressively impair the ability of d-dsRNA to gel electrophoresis to verify that all of the input RNA was deaminated to a similar extent, as indicated by the relatively induce RNAi. 1108 EMBO reports vol. 2 | no. 12 | 2001 scientific reports [α- P]ATP and incubated them under identical conditions as used in the RNAi assay shown in Figure 2B. Incubation of dsRNA in the Drosophila extract gave rise to siRNAs, while no siRNAs were generated from ssRNA (Figure 2D, compare lanes 1 and 7). In this experiment ∼5% of the input dsRNA was proc- essed to siRNAs, while in other experiments up to 15% conver- sion of input dsRNA was observed (data not shown). A progressive decrease in siRNA production was observed to accompany the increase in editing of the d-dsRNA (lanes 2–6), with only background levels detectable at 43% deamination. The reduced levels of siRNA are sufficient to account for the abolition of RNAi at high levels of deamination. siRNAs contain a similar amount of inosine as input RNAs It is possible, at intermediate levels of editing (e.g. 19 % A→I conversion), where siRNA production is reduced only ∼5-fold, that later steps in RNAi may also be affected if the siRNAs include A→I modifications. However, it is possible that the Dicer ribonuclease involved in processing the dsRNA may discriminate against sequences containing I residues due to the altered structure. We therefore carried out an analysis to deter- mine the inosine content of siRNAs generated from d-dsRNA that contained 18% A→I conversion. dsRNA and d-dsRNA were incubated in Drosophila extract for 90 min at 25°C to allow production of siRNAs. The siRNAs and unprocessed RNA from both dsRNA and d-dsRNA were subsequently recovered following denaturing gel electrophoresis. The recovered RNAs were digested with RNase P1 and then subjected to TLC to sepa- Fig. 2. A→I editing progressively inhibits RNAi and production of siRNAs. rate 5′-IMP from 5′-AMP (Figure 3). Prior to incubation in (A) CAT dsRNA was edited to varying degrees (5, 12, 19, 25 and 43% A→I) Drosophila extract, 18% of the A residues in the input d-dsRNA using ADAR2 and analysed by native gel electrophoresis. As the level of were converted to I, while the dsRNA contained no I (lanes 5 editing increased there was a corresponding reduction in their mobility on a native gel (for example, compare lanes 1 and 6). Each deaminated RNA was and 6). The siRNAs produced from d-dsRNA contained 15% A→I relatively homogeneous, as indicated by the relatively tight bands with lower conversion (lane 1), only marginally less than the 18% conver- mobility than unmodified dsRNA. (B) An RNAi assay was performed for sion in the full-length d-dsRNA recovered from the same lane of 90 min where each CAT d-dsRNA was tested for its ability to activate RNAi the denaturing gel (lane 2), and the input d-dsRNA (lane 5). No (lanes 2–6) alongside dsRNA (lane 1) and ssRNA (lane 7). In this assay the control RNA used was ΔKP. As the level of editing increased in the inosine was detectable (<0.8%) in the siRNAs generated from d-dsRNAs, there was a clear reduction in the amount of RNAi (compare lanes the dsRNA or unprocessed dsRNA (lanes 3 and 4). In contrast, 1 and 6). (C) The data from (B) are represented as the CAT/ΔKP ratio relative Zamore et al. (2000) observed ∼3–6% A→I conversion of the ΔKP ratio in the presence of ssRNA. In order to accurately to the CAT/ dsRNA during their RNAi experiments, but <0.7% in the siRNAs. quantitate the amount of target RNA remaining in the presence of highly edited d-dsRNA (lanes 4–6), it was necessary to determine the sum of the two upper This is consistent with our observations if a small proportion of bands that correspond to CAT RNA (marked with an asterisk in lane 6). The their dsRNA were heavily edited and thus unable to generate anomalous mobility of a small proportion of the CAT target RNA is the result siRNAs. The reason for the lower level of endogenous ADAR of partial hybridization of the CAT target RNA with the 200-fold excess of activity in our experiments is not clear. It could be related to the unlabelled CAT d-dsRNA. An inverse correlation exists between the level of editing in d-dsRNA and its ability to induce RNAi. (D) The production of age of the embryos used to prepare the extract (3–6 h as opposed siRNAs from CAT dsRNA and d-dsRNAs was analysed using RNAs labelled to 0–2 h) or to slight differences in the quantity of extract or dura- with [α P]ATP. This assay was performed alongside that shown in (B) using tion of the RNAi assay. Our data show that moderately deami- identical conditions. As the level of editing increased, the production of nated dsRNA can be processed to siRNAs with no strong siRNAs showed a corresponding decrease (compare lanes 1 and 6). The percentage of the input RNA that was converted to siRNAs is shown below discrimination against the generation of siRNAs containing I•U each lane. base pairs. A 15% level of A→I conversion corresponds to an average of 0.8 inosines per siRNA, a level at which ∼55% of the siRNAs would be expected to contain at least one inosine. If perfect Watson–Crick base-pairing between siRNAs and the targeted RNAs is required, the presence of I in the siRNAs will impair RNAi disproportionately more than can be accounted for Editing of RNA reduces the production of siRNAs by the reduced amounts of siRNA. This could be tested directly To test whether hyper-editing directly inhibits the production of by carrying out in vitro RNAi experiments with synthetic siRNAs siRNAs, we labelled trigger RNAs to high specific activity with containing single A–U to I•U substitutions. EMBO reports vol. 2 | no. 12 | 2001 1109 scientific reports A.D.J. Scadden & C.W.J. Smith localization appears likely (Fire et al., 1998). Thus, in some circumstances the two systems may be able to compete directly for access to dsRNA, while elsewhere competition might be determined by which system is first to encounter the dsRNA and thus convert it to a form that cannot be recognized by the other system. METHODS RNAs. All RNAs were prepared by in vitro transcription. The CAT target RNA and the control RNAs were labelled inter- nally with [α- P]UTP (3000 Ci/mmol; Amersham), where Fig. 3. siRNAs contain inosine. dsRNA and d-dsRNA (with 18% A→I) were 50 μCi [α- P]UTP was added per 50 μl transcription incubated in Drosophila extract for 90 min at 25°C. The siRNAs (and reaction. The ‘unlabelled’ trigger RNAs were trace-labelled unprocessed RNA) generated from both dsRNA and d-dsRNA were recovered with [α- P]UTP (3000 Ci/mmol; Amersham) at a specific following gel electrophoresis and analysed for inosine content by RNase P1 activity at least 250-fold lower than that of the target RNA. The digestion followed by TLC. The input d-dsRNA contained 18% A→I conversion (lane 5), while the dsRNA contained no inosine (lane 6). labelled trigger RNAs were labelled internally (on the sense Following recovery from the gel, the unprocessed dsRNA (L) and siRNA strand only) with [α- P]ATP (3000 Ci/mmol; Amersham) to a generated from dsRNA contained no inosine (lanes 3 and 4). In contrast, similar specific activity as the target RNAs. All of the RNAs were unprocessed d-dsRNA (L) contained an equivalent amount of inosine as in initiated using an m G(5′)ppp(5′)G dinucleotide primer. dsRNAs the input d-dsRNA (18% A→I, lane 2). SiRNAs generated from d-dsRNA contained inosine at a level that corresponds to 15% A→I conversion. were prepared by annealing complementary RNAs (Scadden and Smith, 2001). ΔKP RNA (Scadden and Smith, 1997) was synthesized using SP6 RNA polymerase following linearization with BamHI to give a 296-nt transcript. PV 1 RNA (Scadden and Smith, 2001) was Our experiments confirm the predicted biochemical antago- synthesized using T7 RNA polymerase following linearization nism between ADARs and RNAi (Bass, 2000). We used human with BamHI to give a 230-nt transcript. CAT target RNA ADAR2, but similar results would be expected with other (O’Connell and Keller, 1994) was synthesized using T7 RNA poly- ADARs, which show similar maximal levels of ∼50% A→I merase after linearizing the plasmid with PvuII to give an 858-nt conversion upon hyper-editing of long dsRNA substrates (Hough transcript. CAT dsRNA was prepared as described previously and Bass, 2000). Even if there were some differences in the pref- (Scadden and Smith, 2001). VLE RNA comprised nucleotides erences for editing particular adenosines, the overall structural 1383–1813 from the Vg1 cDNA (Pressman-Schwartz et al., 1992). distortions in the dsRNA would be similar, resulting in lack of RNAi assay. The RNAi assay was performed using the conditions processing by Dicer enzymes. At present, it is not clear what described previously (Tuschl et al., 1999; Zamore et al., 2000). physiological consequences result from this antagonism. Expres- The Drosophila extract was prepared from 3–6 h PEL embryos sion of ADARs in various organisms is highest in neural tissues according to the method of Becker et al. (1994). Electrophoresis and also in the developing vulva of C. elegans (L. Tonkin and B. on a 15% polyacrylamide gel enabled the analysis of siRNAs. Bass, personal communication). RNAi in most C. elegans tissues Data were quantitated following phosphorImaging using can be readily achieved by soaking the worms in dsRNA or feeding Imagequant software (Molecular Dynamics). with Escherichia coli strains that express a trigger dsRNA. In Deamination reaction. Deamination reactions were carried out contrast, neurons are relatively resistant (Kamath et al., 2000; as described previously using recombinant human ADAR2 Maeda et al., 2001), and efficient RNAi of neuronally expressed (Scadden and Smith, 2001). The time of the deamination genes has only been achieved by in vivo expression of a herit- reaction was adjusted to achieve varying levels of editing. able inverted repeat gene corresponding to the target gene under Deamination reactions using labelled and unlabelled dsRNAs the control of a strong heat shock-inducible promoter were carried out in parallel to enable quantitation of unlabelled (Tavernarakis et al., 2000). Likewise, injection of dsRNA was d-dsRNAs. To analyse the efficiency of the deamination unable to invoke RNAi in the vulva (Fire et al., 1998). It is reactions, 50 fmol of RNA were digested with RNase P1 and tempting to speculate that the high levels of ADARs in neurons analysed by TLC (Scadden and Smith, 2001). and vulva can explain, at least in part, the relative resistance of genes in these tissues to RNAi (Bass, 2000). This should be ACKNOWLEDGEMENTS readily testable with ADAR knockout strains of C. elegans. Both RNAi and ADARs have been suggested to have roles in We would like to thank Dr Karl Peter Nightingale, Walter Keller antiviral defence, although both systems also have roles in and Anna Git for gifts of reagents, and Brenda Bass for sharing normal development (Emeson and Singh, 2000; Hough and unpublished data and for comments on the manuscript. We Bass, 2000; Grishok et al., 2001; Hutvágner et al., 2001). would also like to thank Mary O’Connell and Witek Filipowicz Mammalian ADARs are usually confined to the nucleus, for comments on the manuscript. This work was supported by a although an interferon inducible ADAR1 isoform is cytoplasmic grant from the Wellcome Trust (052241). A.D.J.S. was also (Patterson and Samuel, 1995). The subcellular localization of the supported by a Research Fellowship from Newnham College, RNAi machinery has not been investigated, but cytoplasmic University of Cambridge. 1110 EMBO reports vol. 2 | no. 12 | 2001 scientific reports Maeda, I., Kohara, Y., Yamamoto, M. and Sugimoto, A. (2001) Large-scale REFERENCES analysis of gene function in Caenorhabditis elegans by high-throughput Bass, B.L. (2000) Double-stranded RNA as a template for gene silencing. RNAi. Curr. Biol., 11, 171–176. Cell, 101, 235–238. Masquida, B. and Westhof, E. (2000) On the wobble G•U and related pairs. Becker, P.B., Tsukiyama, T. and Wu, C. (1994) Chromatin assembly extracts RNA, 6, 9–15. O’Connell, M.A. and Keller, W. (1994) Purification and properties of double- from Drosophila embryos. 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(2001) A role for the RNase III enzyme DCR-1 promiscuously A-to-I edited RNAs. Cell, 106, 465–475. in RNA interference and germ line development in C. elegans. Science, 293, 2269–2271. DOI: 10.1093/embo-reports/kve244 EMBO reports vol. 2 | no. 12 | 2001 1111

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