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Extended target-site specificity for a hammerhead ribozyme
Gene, 113 (1992) 157-163
© 1992 Elsevier Science Publishers B.V. All fights reserved. 0378-1119/92/$05.00
157
GENE 06384
Extended target-site specificity for a hammerhead ribozyme (Catalytic RNA; in vitro mutagenesis; RNA cleavage)
Rhonda Perriman a, Angela Delves b and Wayne L. Getlach a " CSIRO Division of Plant Industry, Canberra, A C T 2601 (Australia) and h Australian National University, Camberra, A C T 2601 (Australia)
Received by J.L. Slightom:9 June 1991 Accepted: 4 July 1991 Received at publishers: 18 January 1992
SUMMARY In vitro mutagenesis has been used to systematically mutate the G U C target site cleaved by a synthetic ribozyme based on the catalytic domain of the satellite RNA of tobacco ringspot virus. Amongst the spectrum of changes, it is found that GUC, UUC, CUC, G U A and G U U targets show equivalent rates of cleavage. An AUC target does not cleave, in contrast to observations from other studies. For a G U G target site, the normal ribozyme cannot induce cleavage, but an alteration of the stem-loop in the catalytic domain leads to the formation of a weakly active ribozyme. Certain double mutations, not previously studied, showed slow but discernable cleavage. This mutational approach shows that general rules for cleavage at NUY triplets for the target site of hammerhead ribozymes should be modified. Not all NUY targets cleave under all circumstances, and there are some targets with nucleotides other than U in the centre position which show significant, discemable cleavage.
INTRODUCTION Autolytic cleavage is a feature of the replication cycle of a number of plant virus satellite RNAs and viroids (Buzayan et al., 1986; Hutchins et al., 1986; Prody et al., 1986; Forster and Symons, 1987; Keese and Symons, 1987; Davies et al., 1990). A consensus model for the RNA structure associated with this cleavage, termed the 'hammerhead'
Correspmtdence to: Dr. R. Perriman, CSIRO Division of Plant Industry, G.P.O. Box 1600, Canberra, ACT 2601 (Australia) Tel. (61-6)2465204; Fax (61-6)2465000.
Abbreviations:bp, base pair(s); CAT,chloramphenicolacetyltransferase; cat, gene (DNA) encoding CAT; DTT, dithiothreitoi; kb, kilobase(s) or
1000bp; nt, nucleotide(s); oligo,oligodeoxyribonucleotide;PAGE, polyacrylamide gel electrophoresis; sTobRV, satellite RNA of tobacco ringspot virus; sLTSV, satellite RNA of lucerne transient streak virus; T,/2, time required for cleavageof half of the available substrate RNA; TCA, trichloroacetic acid; u, unit(s); wt, wildtype; X,X', any standard WatsonCrick nt pair.
model, has been proposed (Forster and Symons, 1987). The cleavage reaction is catalysed in the presence of divalent cations and a relatively neutral pH in producing 2'3' cyclic phosphate and 5' hydroxyl end groups on the RNA products. The cleavage mechanism has been further dissected into bimolecular components in which specific substrate and enzyme RNA molecules (ribozymes) have been defined (Uhlenbeck, 1987; Haseloff and Gerlach, 1988). These ribozymes have subsequently been adapted to successfully target new substrate RNAs (Haseloffand Gerlach, 1988). The structural model on which these ribozymes are designed (see Fig. la) has three basic components: (i)a 22nt highly conserved catalytic domain; (ii)base pairing 5' and 3' of the cleavage target site; (i/i) a cleavage site on the target RNA which generally involves the trinucleotide triplet GUC. To further understand the mechanism of cleavage, the extent of the requirement for these structural components must be determined. This study addresses one of these structural components, the GUC target site.
158 one of the naturally occurring sequences, but rather simply maintained the conserved nt previously determined for the region. Basically, the differences between all these ribozyme sequences are restricted to those nt designated X in Fig. la. Although these nt are designated 'nonconserved', this paper suggests that these regions can affect cleavage site specificity. This paper details a mutagenic analysis of pairwise changes in which substrate/ribozyme base pairing is maintained, along with selected bp mismatches at the target site (see Fig. lb). The results show that new target sites within substrate RNAs can be cleaved in vitro. However, while some target sites have cleavage rates similar to GUC, the majority have markedly slower rates. Evidence is also provided for the modification of previous rules that have been proposed for these cleavage reactions (Koizumi et al., 1989). Certain 'wobble' mismatches between substrate and ribozyme RNAs are also tolerated at this target site. This knowledge is applicable for future in vitro and in vivo design of ribozymes to new target molecules.
At present, all except one of the naturally occurring satellite and viroid RNAs cleave at a GUC target site (Bruening, 1990). The one exception is the minus strand of sLTSV, which cleaves at a GUA target site (Forster and Symons, 1987). Other studies have partly addressed the target-site requirements, although none have tested a systematic series of coordinated nt substitutions at the triplet (Koizumi et al., 1988a,b; 1989; Sheldon and Symons, 1989; Ruffner et al., 1990). To fully interpret the restflts obtained from this study, it is necessary to compare t~.em with these other mutational analyses using related hammerhead ribozyme sequences. All previous studies have based their ribozyme sequences on different naturally occurring self-cleaving satellite and/ or viroid RNAs. Koizumi et al. (1988a,b) used a catalytic domain based on the self-cleaving RNA transcript of the DNA satellite from newt (Epstein and Gall, 1987), while Sheldon and Symons (1989) used the plus-strand sequence from the sLTSV (Forster and Symons, 1987). The catalytic unit used by Ruffner et al. (1990) was not based on any
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b. 1 /'" ~~C~QGGUUU uua,a,Buuucpuc I UCAaCCA~aaUQ,aUUUCACCAaUCCBGG CCI~L~UUUCCCAAAUAACUCUU~ (2) Ribozyme
AAAGG ~ AGUCGGU~CCCACUCAAAGUGGUCAGG~ C C A C A U GAG C'GG A A'U U G'C AG°CG GA
Fig. I. A model for the design of synthetic RNA enzymes based on the autolytic cleavage process from plant virus satellite RNAs and viroids. (a) Structural model. The arrow shows the site of cleavage in the substrate RNA which is effected by the ribozyme. The three structural components are (I) base pairing 5' and 3' of the cleavage site, (2) a conserved catalytic domain containing specific ribonucleotides as shown, (3) a preferred GUC triplet adjacent to the cleavage site on the target RNA. X represents nonconserved nt and the asterisks indicate base pairing. (b) RNA sequences representing transcripts from cloned oligo sequences of (!) substrate - a modified segment of the CAT mRNA (Alton and Vapnek, 1979) containing a GUC cleavage site designated CAT-2, and (2) a ribozyme designed to hybridise and cleave at the specified cleavage site. The cleavage site and mutated nt resulting in the production of specified restriction sites are highlighted. The two sequences were cloned into pOEM3zf- and pGEM3zf+ (Promega, Madison, WI) to allow for the isolation of single-stranded phagemids that were used as templates for the subsequent mutagenesis. Oligos containing multiple redundancies at the GUC/CA target site were designed to test two groups of substrate-ribozymc interactions. There were the (i) XXC/X'X' where X and X' represent any standard Watson-Crick bp and, (ii) GUX/CA. The inset in a dashed box shows detail of the 5 nt around the target site that were altered by mutagenesis to test these. All oligos were synthesised on an Applied Biosystems Model 380A DNA synthesizer. Following mutagenesis, clones were sequenced by double-stranded DNA sequencing using a T7 DNA polymerase kit (Pharmacia, Sweden) to obtain a full library of 35 mutant substrate or ribozyme sequences. In this paper we use the following nomenclature: GUC/CA refers to a 5 ' - G U C target sequence with corresponding base-paired 3'-CA ;n the ribozyme, as shown. Also, X and X' refer to any standard Watson-Crick base pairing.
159 RESULTS AND DISCUSSION
% cleavage 100
(a) Measurement of cleavage rates It is also important to note that among the cleavage rates presented here are the time required for the reactions to achieve 50?/o of product formation (Tl/_,). This was considered valid as it was observed that across the range of experiments, this value was closely related to the initial rate as judged by early time points in the reaction curves. Absolute values for initial reaction rates were not calculated since it is likely that the substrate was undergoing intramolecular folding to produce inactive forms as well as conformations capable of interacting with the ribozyme. This could also account for the observation that none of the reactions went to completion.
(b) GUX target site Alteration of the nonhybridising C residue of the normal G U C target (Fig. 1) to an A or a U in the target site of the G U X / C A pairing produced little reduction in cleavage rates (Fig. 2). The Tl/2 values indicate rates of 8, 20, and 30 rain for GUC, G U A , and G U U target sites, respectively. In contrast, a G U G target site did not exhibit any cleavage when targeted with the standard sTobRV-based ribozyme. Other mutational studies have also shown that the nonhybridising C residue in the target site (Fig. 1) can be altered to an A without marked reduction in cleavage activity (Koizumi et al., 1988a; Sheldon and Symons, 1989; Ruffner et al., 1990). Both Koizumi et al. (1988) and Sheldon and Symons (1989) found little reduction in the rates of cleavage at a G U U target site. Results in this study agree with those of Sheldon and Symons (1989) and Koizumi et al. (1988), with little reduction in cleavage rates when the target site is G U A or G U U , in the presence of the appropriately hybridising ribozyme (Fig. 2). Previous studies have conflicted in their results regarding cleavage of G U G targets. Koizumi et al. (1988a)found that a target site with this sequence could not cleave, whereas Sheldon and Symons (1989)observed considerable cleavage of v G U G substrate, in a unimolecular ribozyme/substrate reaction, both during and after the RNA transcription.
(c) GUG can be induced to cleave The differences observed in G U G cleavage activities with naturally occurring ribozyme sequences may be due simply to the different reaction conditions or the catalytic domains used in each of the studies. As stated earlier, Koizumi et al. (1988a) used a catalytic domain based on the self-cleaving transcript from newt, while Sheldon and Symons (1989) used the sLTSV R N A sequence. We have found that the nature of the catalytic domain can affect G U G cleavage (Fig. 3). When the stem of the catalytic domain used in this
00 no
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1.30
Time (h) GUC/CA TI/2=Bmin --*- GUU/CA TI/2=30min
--t--- GUA/CATl/2=2Omin GUG/CA No traction
Fig. 2. Time course of cleavage reactions after alteration of the nonh~bridising cytosinein the GUC target to an A, U, or Gnt. Percent cleavage is plotted against time. Base pairing has been maintaincd between substrate and ribozyme sequences. RNAs were prepared under the following conditions: 2/41 lineariscd template DNA/40 mM Tris.HCI pH 7.5/ 6 mM MgCI2/2mM spermidine/10 mM NaCI/10 mM DTT/80 u RNasin (Promcga)/! mM ATP, C'IP, GTP/0.25 mM unlabellcd UTP (Pharmacia). Substrate RNAs were transcribed in the presence of 60 pmol of [:¢-3:P]UTP (Bresatech) using 75 u of SP6 RNA polymerasc (Bethcsda Research Labs). Ribozyme transcripts were transcribed in the presence of 6 pmol [~¢-32P]UTPand 100 u ofT7 RNA polymerase(Bethcsda Research Labs). Reactions were incubated at 37~C for 1 to 2 h and then treated with 2 u DNase I (Promcga) to remove the DNA template. Transcript yidds were determined by TCA precipitation and the size and fidelity were checked by denaturing PAGE. Substrate and ribozymeRNAs wcrc mixed and heated to 85~C prior to the addition of buffer (10 mM MgCI2/50mM Tris.HCI pH 7.4). Mg buffer was added, followingquick cooling on ice, and the mixture was immediatelytransferred to 50=C for the reaction. Reactions were terminated by the addition of EDTA to a final concentration of 50 mM followed by cthanol/Na,acetate precipitation. Unless otherwise stated, reaction concentrations were 0.15 pmol substrate and 0.2 pmol ribozymc in a final volumeof 5 pl. Pelleted RNAs were redissolvcd in 100?i, formamidc containing bromophcnol blue and xylcnc cyanol dyes, heated to 90 ~C for 1 rain and rcsolvcd on by PAGE (107~ polyacrylamide/7M urea/40?~, formamidc) using the following buffer: 180 mM Tris.borate pH 8.3/4 mM EDTA. Radioactive products were excised and counted to determine the extent and rate of cleavage. It should be noted that all cleavage reactions were repeated a minimum of three times with the average rates of product formation plotted using Harvard GraphicsTM (Software PublishingCorp, Mountain View,CA) as shown. For each substrate/ribozyme cleavage reaction tested, the standard GUC substrate/CA ribozyme was done in parallel. The cleavage rates are presented here as Tz/2. study was doubled in length (i.e., a duplication of the sTobRV stem) and the terminating loop maintained, the G U G target site was then observed to cleave, albeit at a markedly reduced rate (Fig. 3). It may be that, in placing a G in the 3' position of the cleavage site, the most 5' C of the catalytic domain may now hybridise to this nt. This, in turn, could alter the structure of the standard sTobRV catalytic domain, providing an alternate nonactive conformation. The catalytic domain used by Sheldon and Symons (1989) has 26 nt, three more than the sTobRV-based domain used in our study, including an extra bp in the stem. This may confer the necessary
160 1
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7= c l e a v a g e
7
100
GUG XCA
80
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60 40 20,
Rz
XRz
5'P
3'P
Fig. 3. Autoradiograph showing cleavage of a substrate containing a GUG target site, when targeted with a ribozyme containing a duplication of the stem. That is, instead of the normal 4 bp terminated with a loop (Fig. lb), the extended stem ribozyme has an 8-bp region. Cleavage re. actions and PAGE were carried out as described in Fig. 2. Lanes: !, GUC substrate; 2, CA ribozyme; 3, GUC/CA cleavage reaction; 4, GUG sub. strate; 5, GUG/CA no reaction', 6, CA extended stem ribozyme; 7, GUG/ extended stem CA cleavage reaction. The RNA species are as follows: S, substrate; Rz, ribozyme; XRz, extended stem ribozymv; 5'P, product RNA 5' of the target site; 3'P, prodt~et RNA 3' of the target site.
stability to the catalytic domain to tolerate a G:C pairing in the third position at the target site or alternatively, the additional bp in the ribozyme used by Sheldon and Symons (1989) and the extended stem ribozyme used in this study may, in fact, inhibit that G:C pairing. This could account for these observations that some catalytic domains can cleave at a GUG target site while others cannot. However, this explanation is complicated by the observation that the ribozyme used by Koizumi et al. (1988a) cannot cleave a GUG target site and yet it has the same length stem as the catalytic domain used by Sheldon and Symons (1989). A difference lies in the fact that the catalytic domain used by Koizumi et al. (1989) is not terminated with a loop. Recent comparisons between two such forms of ribozymes have repeatedly shown that a catalytic domain containing a looped stem induces more efficient cleavage in vitro compared to an equivalent catalytic domain that is not terminated with a loop (Ruffner et al., 1989; Chang et al., 1990; Sarver et al., 1990). The terminating loop is perhaps providing the ribozyme with a greater
0.00 0.10 0.20 o.ao 0.40 0.~0 l.oo l.lo 1.20 l.ao 1.40 ,.ao 2.00 T i m e (h) GUC/CA T l / 2 = f l m i n
--t....-- UUC/AA T l / 2 = 1 5 m i n
CUC/GA T l / 2 = 2 5 m i n
-~'-
AUC/UA No r e a c t i o n
Fig. 4. Time-course reactions after alteration of the G nt in the GUC target site to each of the three other ribonucleotides. Compensatory changes were incorporated in the ribozyme sequence so that full basepairing between substrate and ribozyme was maintained, i.e., XUC/CA where XUC represents the target site on the substrat¢ and X'A is the corresponding base pairing on the ribozyme, as described in Fig. 1, Cleavage reactions and subsequent analyses were carried out as described in Fig. 2.
ability to fold rapidly into the correct conformation so that efficient cleavage is produced. Thus, the sTobRV-based ribozyme with an extended stem and loop might have the structural stability to induce cleavage at a GUG cleavage site. (d) XUC target site Results for the XUC mutants are shown in Fig. 4. Cleavage was observed for GUC (i.e., wt), CUC and UUC target sites when substrate/ribozyme base-pairing was maintained. The T~/2 values for the substrate/ribozyme pairs UUC/AA and CUC/GA were 15 and 25 rain, respectively. No cleavage of an AUC substrate was observed. These results, therefore, showed efficient cleavage of GUC, CUC and UUC target sites when substrate/ ribozym¢ hybridisation is fully maintained. Contrary to other reports (Koizumi et al., 1988a; Ruffner et ai., 1990), an AUC target site did not cleave. One explanation for this difference could be the ability of the substrate and/or ribozyme in our system to form an alternative structure which impedes cleavage. Heus et al. (1990) suggested that any conformation of the ribozyme and/or substrate RNA that interferes with complex formation could decrease the apparent rate of cleavage. Further to this, Fedor and Uhlenbeck (1990) have attributed variable cleavage rates to possible secondary structures in a given substrate RNA. This may also explain the apparent discrepancies between different systems which have been reported and suggests that a 'general' rule regarding the target site cleavage rates for ribozymes may not apply. What can be drawn from our results, however, is that if a U:A is the central bp of the target site (i.e., a U in the substrate and an A in the hybridising position on the ribozyme), a relatively high degree of flexibility is tolerated in the other two positions of the
161 cleavage I00
(a) (b) (c)
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(a) (b) (c)
(a) (b) (c)
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ACC CCC GCC UCC ACC UG +UG CCC GG +GG GCC CG +CG UCC AG +AG
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.
.
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~:
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AAC/UU TlO,~.=4h
~
~
Rz
Sub
LIAC/AO Tl0,~,=l.fh
(;AC/CU No reaction
Fig. 5. A time course involving XAC/X'U substrate-ribozyme interactions. The GUC/CA target site cleavage rate is also illustrated as a control. T.j.,,.. the time required for the reaction to achieve 10% of product formation.
triplet. This is consistent with observations made with single nt changes by Koizumi et al. (1988a).
(e) Site-specific cleavage occurs with changes to the middle residue of the GUC triplet Although a U residue in the central position of the triplet appears necessary for efficient cleavage, we have observed that certain XAC and XCC triplets can also be cleaved. Results in Fig. 5 show that certain XAC substrates, in the presence of the appropriate X'U ribozyme, will cleave specifically, although at a markedly reduced rate. While double mutations AAC/UU, CAC/GU and UAC/AU all exhibited cleavage, the GAC substrate did not cleave. T~/2 values for the three constructs capable of cleaving were not obtained due to the slow rate of reaction. However, T~o% values were 4, 2, and 1.5 h, respectively. The corresponding Tioo. for GUC/CA was less than 5 rain in a series of experiments. Target sites containing an XCC triplet also cleave, but rates were too slow to get any indication of significant activity (Fig. 6). Cleavage was only observable following several hours of incubation of concentrated ribozyme/ substrate mixes (i.e., 1.5 pmol substrate and 2pmol ribozyme in a 5-#1 reaction). Within the XCC mutants, the ACC and the UCC target sites were most efficiently cleaved. These are double mutants from the normal GUC target and have not been studied previously. Their increased rate compared to CCC and GCC target sites may be due to greater flexibility afforded by weaker A:U pairing compared with stronger G:C pairing. This is supported by the observation that those XCC target sites comprised exclusively of G:C pairing were very much slower, if exhibiting cleavage at all, than those containing an A:U pair. The ACC/UG and UCC/AG reactions showed greater activity than CCC/GG and GCC/CG. This was not the case for the XGC/X'C substrate/ribozyme interactions which showed no observ-
5'P 3'P Fig. 6. Autoradiograph showing cleavage reactions involving XCC mutant substrate RNAs mixed with the appropriate mutant ribozyme so that base pairing between the two sequences was maintained, i.e., XCC/X'G. The 5-pl reaction volumes contained 1.5 pmoi of substrate and/or 2 pmol of ribozyme, incubated individually or together for 4 h under standard reaction conditions as described in Fig. 2. For each of the XCC/X'G combinations, the lanes show: a, XCC substrate incubated alone; b, X'G ribozyme incubated alone; e, reaction products for XCC substrate and/ X'G ribozyme incubated together. Cleavage reactions and PAGE were carried out as described in Fig. 2. The RNA species are as follows: Sub, substrate RNA; Rz, ribozyme; 5'P, cleavage product representing the sequence 5' ofthe active site; 3' P, 3' cleavage product. The high-Mr band labelled 'Corn' is most likely to be nondenatured substrate/ribozyme complex.
able site-specific cleavage. It indicates that a G as the central residue in the target-site triplet is not tolerated. Only GGC had been tested in previous work and was found not to cleave (Ruffner et al., 1990).
(f) G:U base pairing is tolerated at the target site Non Watson-Crick alignments (termed wobble base pairing) can occur in nucleic acid hybrids. In particular, a G:U pairing has been found to approximate an A:U pairing in stabilising efficiency (Saenger, 1984). It has structural similarities to Watson-Crick base-pairing and therefore does not disrupt the double helix, causing only a slight 'bulge' in the sugar-phosphate backbone (Saenger, 1984). For this reason G:U mismatches were tested in the target site/ribozyme interaction. It was found that cleavage reactions can occur under these conditions (Table I). Two target sites, GUC and UUC, were chosen because they can be cleaved efficiently when normal base pairing is mainrained. This made the assays of relative rates more accurate and also enabled us to observe the effect of wobble pairings in both hybridising positions. In the case of the GUC target, site-specific cleavage was only observed for the GUC/CA normal and GUC/CG
162 TABLE I Effect of G:U 'wobble' base pairing at the target site on cleavage rates Substrate"' Ribozyme
Relative activity (%) h
GUC •
Substrate"' Ribozyme
Relative activity i o~ / O I~b
UUC
.
CA
66 6;,
100 70
U,
120 21
0 o
X6
0 o
" Two target sites. UUC and GUC were incubated with ribozymeswhich introduced G:U base pairing in either and/or both hybridisingpositions. Dots indicate potential base pairing between the substrate target site and four ribozymes. The substrat¢ sequence is shown at the top of each column with the ribozymes listed underneath. h Relativeactivityrepresents the extent of cleavage after 1 h when GUC/ CA is expressed as 100%. Reaction conditions were as outlined in Fig. 2. wobble combinations. Therefore, in this instance a wobble mismatch was tolerated in the second position of the target site but not in the first. However, upon targeting U U C substrate, only U U C / A A normal and U U C / G A wobble combinations showed cleavage. In this case a wobble mismatch is tolerated in the first position but not in the second. G U C / U G and U U C / G G did not cleave indicating that a wobble mismatch at both positions was not tolerated (Table I). The G U C / C G reaction was slower than the correct GUC/CA pairing although rates were still relatively fast (Table I). In comparison, the U U C / G A pairing is markedly slower than its Watson-Crick counterpart. The Tie,. for U U C / G A was 40 rain, whereas the U U C / A A is < 5 rain. Therefore, it appears that wobble base pairing is permissible in some, but not all, positions in the UUC and G U C target sites studied (Table I). Both of the wobble mismatches which showed cleavage had slower cleavage rates when compared with their Watson-Crick counterparts. The G U C / C G pairing was slower than the correct G U C / C A pairing although rates were still relatively fast. Presumably, as the stability of the hybrid is similar, the effect on cleavage is a result of the slight structural deviation from the standard bp geometry. These results are different to those obtained previously for permissible G:U bp at the target site. Sheldon and Symons (1989) found in their system that a G:U bp is tolerated with the G at the first position of the G U C target triplet (i.e., a G in the substrate and a U in the ribozyme) and not in the second. Once again, it is likely that other components contributing to the overall structure are again affecting the specificity with which these ribozymes can induce cleavage. Regarding the other potential wobble base-pairing situation, the U U C / G A pairing is markedly slower than the normal U U C / A A reaction. Such an interaction has not
been studied previously. Perhaps the absence of relatively strong G:C pairing in the U U C target/ribozyme interaction enhances the effect of the wobble pairing. This could explain why the G U C substrate can tolerate a wobble in the second position but U U C cannot. The bp immediately 5' of the target site is C:G. Perhaps this allows U U C to wobble in the first position but not the second. Thus it seems that U:A pairing can be replaced by a U:G as long as there is a G:C pair immediately to the 5' side of it. However, a G:C pairing could not be replaced by a G:U in either of the two cases examined (Table I).
(g) Conclusions Overall, it now appears that a degree of flexibility can be tolerated in the target site of an RNA substrate in the ribozyme/substrate interactions based on the hammerhead cleavage domains. Even certain wobble base pairings can be accommodated. In all but two cases tested a U as the central residue of the target-site triplet permits efficient sitespecific cleavage. An A or C in this position, with certain base pairings in the first position can also undergo cleavage. These results are summarised in Table II. This work indicates that the general ruling of an N U Y target site (Koizumi et al., 1989) may not hold. Instead, a consensus target rule may not be possible as different target sequences TABLE II Summary of the reaction rates of target sites altered by she specific mutagenesis with base pairing maintained between substrate and ribozyme in all cases Efficient cleavage"
Lower efficiencyh
No activity~
Target Relative Target Relative Target Relative sited activity(°.8)~ site'~ activity(%)~ sited activity(',<.,)~ GUC GUA GUU UUC CUC
100 93 68 120 95
CAC UAC AAC CCC UCC ACC GCC
4 6 3
GAC GUG AUC CGC GGC AGC UGC
NR NR NR NR NR NR NR
" Substratetarget site triplets showingefficientrates of cleavage when the wt target, GUC, is assigned as 100 (i.e., other rates ale expressed relative to GUC). Substrate target site triplets showing low level rates of cleavage when compared to the wt GUC triplet. As the XCC targets listed here, showed cleavage under conditions of high concentration only, they have not been assigned relative rates. ~" Substrate target site triplets showing no discernable cleavage. d Mutant target sites on substrate RNAs. Each target site was combined with a mutant ribozyme such that full base pairing was maintained. Relative activity of each mutant target site after 1 h incubation under standard reaction conditions as outlined in Fig. 2. The wt target GUC = 100, with rates for other target sites expressed relative to this. NR, no reaction.
163 show different cleavage activities according to their own sequence context along with those of the corresponding ribozyme. As structural models of the catalytic domain and cleavage process are developed, these results will need to be accommodated within these observations. This work also provides valuable information for future manipulations involving ribozymes by indicating a number of new and unique target sites as potential cleavage sites for use both in vitro and in vivo.
ACKNOWLEDGEMENTS
The authors wish to thank Lynda Graf for the synthesis ofthe oligos. Support for part of this work was provided under the Generic Technology component of the Australian Government Industry Research and Development Act, 1986.
REFERENCES Alton, N.K. and Vapnek, D.: Nucleotide sequence analysis of the chloramphenicol resistance transposon, Tn9. Nature 282 (1979) 864-869. Bruening, G.: Compilation of self-cleaving sequences from plant virus satellite RNAs and other sources. Methods Enzymoi. 180 (1990) 546-558. Buzayan, J.M., Gerlach, W.L. and Bruening, G.: Nonenzymatic cleavage and ligation of RNAs complementary to a plant virus satellite RNA. Nature 323 (1986) 349-353. Chang, P.S., Cantin, E.M., Zaia, LA., Ladne, P.A., Stephens, D.A., Sarver, N. and Rossi, JJ.: Ribozyme-mediated site-specific cleavage oft~e HIV-! genome. Clin. Biotechnol. 2 (1990) 23-31. Davies, C., Haseloff, J. and Symons, R.H.: Structure, stir-cleavage and replication of two viroid-like satellite RNAs of subterranean clover mottle virus. Virology 177 (1990) 216-224. Epstein, L.M. and Gall, J.G.: Self-cleaving transcripts of satellite DNA from the newt. Cell 48 (1987) 535-543. Fedor, J.J. and Uhlenbeck, O.C.: Substrate sequence effects on 'ham-
merhead' RNA catalytic efficiency. Proc. Natl. Acad. Sci. USA 87 (1990) 1668-1672. Forster, A.C. and Symons, R.H.: Self-cleavage of plus and minus RNAs ofa virusoid and a structural model for the active sites. Cell 49 (1987) 211-220. Haseloff, J. and Gerlach, W.L.: Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature 334 (1988) 585591. Heus, H.A., Uhlenbeck, O.C. and Pardi, A.: Sequence dependent structural variations of hammerhead RNA enzymes. Nucleic Acids Res. 18 (1990) 1103-1108. Hutchins, C.J., Rathjen, P.J., Forster, A.C. and Symons, R.H.: Selfcleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res. 14 (1986) 3627-3640. Keese, P. and Symons, R.H.: The structure of viroids and virusoids. In: Semancik, J.S. (Ed.), Viroids and Viroid-like Pathogens. CRC Press, Boca Raton, FL, 1987, pp. 1-47. Koizumi, M., lwai, S. and Ohtsuka, E.: Construction of a series of selfcleaving RNA duplexes using synthetic 21-reefs. FEBS Lett. 228 (1988a) 228-230. Koizumi, M., lwai, S. and Ohtsuka, E.: Cleavage of specific sites of RNA by designed ribozymes. FEBS Lett. 239 (1988b) 285-288. Koizumi, M., Hayase, Y., lwai, S., Kamiya, H., Inoue, H. and Ohtsuka, E.: Design of RNA enzymes distinguishing a single base mutation in RNA. Nucleic Acids Res. 17 (1989) 7059-7071. Kunkel, T.A., Roberts, .I.D. and Zakour, R.A.: Rapid and efficient site specific mutagenesis without phenotypic selection. Methods Enzymol. 154 (1987) 367-382. Prody, G.A., Bakos, J.T., Buzayan, J.M., Schneider, I.R. and Bruening, G.: Autolytic processing ofdimeric plant virus satellite RNA. Science 231 (1986) 1577-1580. Ruffner, D.E., Dahm, S.C. and Uhlenbeck, O.C.: Studies on the hammerhead RNA self-cleaving domain. Gene 82 (1989) 31-41. Ruffner, D.E., Stormo, G.D. and Uhlenbeck, O.C.: Sequence requirements of the hammerhead RNA self-cleavage react/on. Biochemistry 29 (1990) 10695-10702. Saenger, W.: Principles of Nucleic Acid Structure. Springer-Verlag, New York, 1984, pp. 149-158 and 334-337. Sarver, N., Cantin, E.M., Chang, P.S., Zaia, J.A., Ladne, P.A., Stephens, D.A. and Rossi, J.J.: Ribozymes as potential anti-HIV-1 therapeutic agents. Science 247 (1990) 1222-1225. Sheldon, C.C. and Symons, R.H.: Mutagenesis analysis of a self-cleaving RNA. Nucleic Acids Res. 17 (1989) 5556-5562. Uhlenbeck, O.C.: A small catalytic oligoribonucleotide. Nature 328 (1987) 596-600.
© 1992 Elsevier Science Publishers B.V. All fights reserved. 0378-1119/92/$05.00
157
GENE 06384
Extended target-site specificity for a hammerhead ribozyme (Catalytic RNA; in vitro mutagenesis; RNA cleavage)
Rhonda Perriman a, Angela Delves b and Wayne L. Getlach a " CSIRO Division of Plant Industry, Canberra, A C T 2601 (Australia) and h Australian National University, Camberra, A C T 2601 (Australia)
Received by J.L. Slightom:9 June 1991 Accepted: 4 July 1991 Received at publishers: 18 January 1992
SUMMARY In vitro mutagenesis has been used to systematically mutate the G U C target site cleaved by a synthetic ribozyme based on the catalytic domain of the satellite RNA of tobacco ringspot virus. Amongst the spectrum of changes, it is found that GUC, UUC, CUC, G U A and G U U targets show equivalent rates of cleavage. An AUC target does not cleave, in contrast to observations from other studies. For a G U G target site, the normal ribozyme cannot induce cleavage, but an alteration of the stem-loop in the catalytic domain leads to the formation of a weakly active ribozyme. Certain double mutations, not previously studied, showed slow but discernable cleavage. This mutational approach shows that general rules for cleavage at NUY triplets for the target site of hammerhead ribozymes should be modified. Not all NUY targets cleave under all circumstances, and there are some targets with nucleotides other than U in the centre position which show significant, discemable cleavage.
INTRODUCTION Autolytic cleavage is a feature of the replication cycle of a number of plant virus satellite RNAs and viroids (Buzayan et al., 1986; Hutchins et al., 1986; Prody et al., 1986; Forster and Symons, 1987; Keese and Symons, 1987; Davies et al., 1990). A consensus model for the RNA structure associated with this cleavage, termed the 'hammerhead'
Correspmtdence to: Dr. R. Perriman, CSIRO Division of Plant Industry, G.P.O. Box 1600, Canberra, ACT 2601 (Australia) Tel. (61-6)2465204; Fax (61-6)2465000.
Abbreviations:bp, base pair(s); CAT,chloramphenicolacetyltransferase; cat, gene (DNA) encoding CAT; DTT, dithiothreitoi; kb, kilobase(s) or
1000bp; nt, nucleotide(s); oligo,oligodeoxyribonucleotide;PAGE, polyacrylamide gel electrophoresis; sTobRV, satellite RNA of tobacco ringspot virus; sLTSV, satellite RNA of lucerne transient streak virus; T,/2, time required for cleavageof half of the available substrate RNA; TCA, trichloroacetic acid; u, unit(s); wt, wildtype; X,X', any standard WatsonCrick nt pair.
model, has been proposed (Forster and Symons, 1987). The cleavage reaction is catalysed in the presence of divalent cations and a relatively neutral pH in producing 2'3' cyclic phosphate and 5' hydroxyl end groups on the RNA products. The cleavage mechanism has been further dissected into bimolecular components in which specific substrate and enzyme RNA molecules (ribozymes) have been defined (Uhlenbeck, 1987; Haseloff and Gerlach, 1988). These ribozymes have subsequently been adapted to successfully target new substrate RNAs (Haseloffand Gerlach, 1988). The structural model on which these ribozymes are designed (see Fig. la) has three basic components: (i)a 22nt highly conserved catalytic domain; (ii)base pairing 5' and 3' of the cleavage target site; (i/i) a cleavage site on the target RNA which generally involves the trinucleotide triplet GUC. To further understand the mechanism of cleavage, the extent of the requirement for these structural components must be determined. This study addresses one of these structural components, the GUC target site.
158 one of the naturally occurring sequences, but rather simply maintained the conserved nt previously determined for the region. Basically, the differences between all these ribozyme sequences are restricted to those nt designated X in Fig. la. Although these nt are designated 'nonconserved', this paper suggests that these regions can affect cleavage site specificity. This paper details a mutagenic analysis of pairwise changes in which substrate/ribozyme base pairing is maintained, along with selected bp mismatches at the target site (see Fig. lb). The results show that new target sites within substrate RNAs can be cleaved in vitro. However, while some target sites have cleavage rates similar to GUC, the majority have markedly slower rates. Evidence is also provided for the modification of previous rules that have been proposed for these cleavage reactions (Koizumi et al., 1989). Certain 'wobble' mismatches between substrate and ribozyme RNAs are also tolerated at this target site. This knowledge is applicable for future in vitro and in vivo design of ribozymes to new target molecules.
At present, all except one of the naturally occurring satellite and viroid RNAs cleave at a GUC target site (Bruening, 1990). The one exception is the minus strand of sLTSV, which cleaves at a GUA target site (Forster and Symons, 1987). Other studies have partly addressed the target-site requirements, although none have tested a systematic series of coordinated nt substitutions at the triplet (Koizumi et al., 1988a,b; 1989; Sheldon and Symons, 1989; Ruffner et al., 1990). To fully interpret the restflts obtained from this study, it is necessary to compare t~.em with these other mutational analyses using related hammerhead ribozyme sequences. All previous studies have based their ribozyme sequences on different naturally occurring self-cleaving satellite and/ or viroid RNAs. Koizumi et al. (1988a,b) used a catalytic domain based on the self-cleaving RNA transcript of the DNA satellite from newt (Epstein and Gall, 1987), while Sheldon and Symons (1989) used the plus-strand sequence from the sLTSV (Forster and Symons, 1987). The catalytic unit used by Ruffner et al. (1990) was not based on any
CAT-2
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CLEAVAGE SITE
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b. 1 /'" ~~C~QGGUUU uua,a,Buuucpuc I UCAaCCA~aaUQ,aUUUCACCAaUCCBGG CCI~L~UUUCCCAAAUAACUCUU~ (2) Ribozyme
AAAGG ~ AGUCGGU~CCCACUCAAAGUGGUCAGG~ C C A C A U GAG C'GG A A'U U G'C AG°CG GA
Fig. I. A model for the design of synthetic RNA enzymes based on the autolytic cleavage process from plant virus satellite RNAs and viroids. (a) Structural model. The arrow shows the site of cleavage in the substrate RNA which is effected by the ribozyme. The three structural components are (I) base pairing 5' and 3' of the cleavage site, (2) a conserved catalytic domain containing specific ribonucleotides as shown, (3) a preferred GUC triplet adjacent to the cleavage site on the target RNA. X represents nonconserved nt and the asterisks indicate base pairing. (b) RNA sequences representing transcripts from cloned oligo sequences of (!) substrate - a modified segment of the CAT mRNA (Alton and Vapnek, 1979) containing a GUC cleavage site designated CAT-2, and (2) a ribozyme designed to hybridise and cleave at the specified cleavage site. The cleavage site and mutated nt resulting in the production of specified restriction sites are highlighted. The two sequences were cloned into pOEM3zf- and pGEM3zf+ (Promega, Madison, WI) to allow for the isolation of single-stranded phagemids that were used as templates for the subsequent mutagenesis. Oligos containing multiple redundancies at the GUC/CA target site were designed to test two groups of substrate-ribozymc interactions. There were the (i) XXC/X'X' where X and X' represent any standard Watson-Crick bp and, (ii) GUX/CA. The inset in a dashed box shows detail of the 5 nt around the target site that were altered by mutagenesis to test these. All oligos were synthesised on an Applied Biosystems Model 380A DNA synthesizer. Following mutagenesis, clones were sequenced by double-stranded DNA sequencing using a T7 DNA polymerase kit (Pharmacia, Sweden) to obtain a full library of 35 mutant substrate or ribozyme sequences. In this paper we use the following nomenclature: GUC/CA refers to a 5 ' - G U C target sequence with corresponding base-paired 3'-CA ;n the ribozyme, as shown. Also, X and X' refer to any standard Watson-Crick base pairing.
159 RESULTS AND DISCUSSION
% cleavage 100
(a) Measurement of cleavage rates It is also important to note that among the cleavage rates presented here are the time required for the reactions to achieve 50?/o of product formation (Tl/_,). This was considered valid as it was observed that across the range of experiments, this value was closely related to the initial rate as judged by early time points in the reaction curves. Absolute values for initial reaction rates were not calculated since it is likely that the substrate was undergoing intramolecular folding to produce inactive forms as well as conformations capable of interacting with the ribozyme. This could also account for the observation that none of the reactions went to completion.
(b) GUX target site Alteration of the nonhybridising C residue of the normal G U C target (Fig. 1) to an A or a U in the target site of the G U X / C A pairing produced little reduction in cleavage rates (Fig. 2). The Tl/2 values indicate rates of 8, 20, and 30 rain for GUC, G U A , and G U U target sites, respectively. In contrast, a G U G target site did not exhibit any cleavage when targeted with the standard sTobRV-based ribozyme. Other mutational studies have also shown that the nonhybridising C residue in the target site (Fig. 1) can be altered to an A without marked reduction in cleavage activity (Koizumi et al., 1988a; Sheldon and Symons, 1989; Ruffner et al., 1990). Both Koizumi et al. (1988) and Sheldon and Symons (1989) found little reduction in the rates of cleavage at a G U U target site. Results in this study agree with those of Sheldon and Symons (1989) and Koizumi et al. (1988), with little reduction in cleavage rates when the target site is G U A or G U U , in the presence of the appropriately hybridising ribozyme (Fig. 2). Previous studies have conflicted in their results regarding cleavage of G U G targets. Koizumi et al. (1988a)found that a target site with this sequence could not cleave, whereas Sheldon and Symons (1989)observed considerable cleavage of v G U G substrate, in a unimolecular ribozyme/substrate reaction, both during and after the RNA transcription.
(c) GUG can be induced to cleave The differences observed in G U G cleavage activities with naturally occurring ribozyme sequences may be due simply to the different reaction conditions or the catalytic domains used in each of the studies. As stated earlier, Koizumi et al. (1988a) used a catalytic domain based on the self-cleaving transcript from newt, while Sheldon and Symons (1989) used the sLTSV R N A sequence. We have found that the nature of the catalytic domain can affect G U G cleavage (Fig. 3). When the stem of the catalytic domain used in this
00 no
40 2o o 0.00
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~ 0.20
0.30
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0 ,50
1.00
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1.30
Time (h) GUC/CA TI/2=Bmin --*- GUU/CA TI/2=30min
--t--- GUA/CATl/2=2Omin GUG/CA No traction
Fig. 2. Time course of cleavage reactions after alteration of the nonh~bridising cytosinein the GUC target to an A, U, or Gnt. Percent cleavage is plotted against time. Base pairing has been maintaincd between substrate and ribozyme sequences. RNAs were prepared under the following conditions: 2/41 lineariscd template DNA/40 mM Tris.HCI pH 7.5/ 6 mM MgCI2/2mM spermidine/10 mM NaCI/10 mM DTT/80 u RNasin (Promcga)/! mM ATP, C'IP, GTP/0.25 mM unlabellcd UTP (Pharmacia). Substrate RNAs were transcribed in the presence of 60 pmol of [:¢-3:P]UTP (Bresatech) using 75 u of SP6 RNA polymerasc (Bethcsda Research Labs). Ribozyme transcripts were transcribed in the presence of 6 pmol [~¢-32P]UTPand 100 u ofT7 RNA polymerase(Bethcsda Research Labs). Reactions were incubated at 37~C for 1 to 2 h and then treated with 2 u DNase I (Promcga) to remove the DNA template. Transcript yidds were determined by TCA precipitation and the size and fidelity were checked by denaturing PAGE. Substrate and ribozymeRNAs wcrc mixed and heated to 85~C prior to the addition of buffer (10 mM MgCI2/50mM Tris.HCI pH 7.4). Mg buffer was added, followingquick cooling on ice, and the mixture was immediatelytransferred to 50=C for the reaction. Reactions were terminated by the addition of EDTA to a final concentration of 50 mM followed by cthanol/Na,acetate precipitation. Unless otherwise stated, reaction concentrations were 0.15 pmol substrate and 0.2 pmol ribozymc in a final volumeof 5 pl. Pelleted RNAs were redissolvcd in 100?i, formamidc containing bromophcnol blue and xylcnc cyanol dyes, heated to 90 ~C for 1 rain and rcsolvcd on by PAGE (107~ polyacrylamide/7M urea/40?~, formamidc) using the following buffer: 180 mM Tris.borate pH 8.3/4 mM EDTA. Radioactive products were excised and counted to determine the extent and rate of cleavage. It should be noted that all cleavage reactions were repeated a minimum of three times with the average rates of product formation plotted using Harvard GraphicsTM (Software PublishingCorp, Mountain View,CA) as shown. For each substrate/ribozyme cleavage reaction tested, the standard GUC substrate/CA ribozyme was done in parallel. The cleavage rates are presented here as Tz/2. study was doubled in length (i.e., a duplication of the sTobRV stem) and the terminating loop maintained, the G U G target site was then observed to cleave, albeit at a markedly reduced rate (Fig. 3). It may be that, in placing a G in the 3' position of the cleavage site, the most 5' C of the catalytic domain may now hybridise to this nt. This, in turn, could alter the structure of the standard sTobRV catalytic domain, providing an alternate nonactive conformation. The catalytic domain used by Sheldon and Symons (1989) has 26 nt, three more than the sTobRV-based domain used in our study, including an extra bp in the stem. This may confer the necessary
160 1
GUC
2
3
4
CA
GUC CA
GUG
5
GUG CA
6
XCA
7= c l e a v a g e
7
100
GUG XCA
80
_ _ - -
,
60 40 20,
Rz
XRz
5'P
3'P
Fig. 3. Autoradiograph showing cleavage of a substrate containing a GUG target site, when targeted with a ribozyme containing a duplication of the stem. That is, instead of the normal 4 bp terminated with a loop (Fig. lb), the extended stem ribozyme has an 8-bp region. Cleavage re. actions and PAGE were carried out as described in Fig. 2. Lanes: !, GUC substrate; 2, CA ribozyme; 3, GUC/CA cleavage reaction; 4, GUG sub. strate; 5, GUG/CA no reaction', 6, CA extended stem ribozyme; 7, GUG/ extended stem CA cleavage reaction. The RNA species are as follows: S, substrate; Rz, ribozyme; XRz, extended stem ribozymv; 5'P, product RNA 5' of the target site; 3'P, prodt~et RNA 3' of the target site.
stability to the catalytic domain to tolerate a G:C pairing in the third position at the target site or alternatively, the additional bp in the ribozyme used by Sheldon and Symons (1989) and the extended stem ribozyme used in this study may, in fact, inhibit that G:C pairing. This could account for these observations that some catalytic domains can cleave at a GUG target site while others cannot. However, this explanation is complicated by the observation that the ribozyme used by Koizumi et al. (1988a) cannot cleave a GUG target site and yet it has the same length stem as the catalytic domain used by Sheldon and Symons (1989). A difference lies in the fact that the catalytic domain used by Koizumi et al. (1989) is not terminated with a loop. Recent comparisons between two such forms of ribozymes have repeatedly shown that a catalytic domain containing a looped stem induces more efficient cleavage in vitro compared to an equivalent catalytic domain that is not terminated with a loop (Ruffner et al., 1989; Chang et al., 1990; Sarver et al., 1990). The terminating loop is perhaps providing the ribozyme with a greater
0.00 0.10 0.20 o.ao 0.40 0.~0 l.oo l.lo 1.20 l.ao 1.40 ,.ao 2.00 T i m e (h) GUC/CA T l / 2 = f l m i n
--t....-- UUC/AA T l / 2 = 1 5 m i n
CUC/GA T l / 2 = 2 5 m i n
-~'-
AUC/UA No r e a c t i o n
Fig. 4. Time-course reactions after alteration of the G nt in the GUC target site to each of the three other ribonucleotides. Compensatory changes were incorporated in the ribozyme sequence so that full basepairing between substrate and ribozyme was maintained, i.e., XUC/CA where XUC represents the target site on the substrat¢ and X'A is the corresponding base pairing on the ribozyme, as described in Fig. 1, Cleavage reactions and subsequent analyses were carried out as described in Fig. 2.
ability to fold rapidly into the correct conformation so that efficient cleavage is produced. Thus, the sTobRV-based ribozyme with an extended stem and loop might have the structural stability to induce cleavage at a GUG cleavage site. (d) XUC target site Results for the XUC mutants are shown in Fig. 4. Cleavage was observed for GUC (i.e., wt), CUC and UUC target sites when substrate/ribozyme base-pairing was maintained. The T~/2 values for the substrate/ribozyme pairs UUC/AA and CUC/GA were 15 and 25 rain, respectively. No cleavage of an AUC substrate was observed. These results, therefore, showed efficient cleavage of GUC, CUC and UUC target sites when substrate/ ribozym¢ hybridisation is fully maintained. Contrary to other reports (Koizumi et al., 1988a; Ruffner et ai., 1990), an AUC target site did not cleave. One explanation for this difference could be the ability of the substrate and/or ribozyme in our system to form an alternative structure which impedes cleavage. Heus et al. (1990) suggested that any conformation of the ribozyme and/or substrate RNA that interferes with complex formation could decrease the apparent rate of cleavage. Further to this, Fedor and Uhlenbeck (1990) have attributed variable cleavage rates to possible secondary structures in a given substrate RNA. This may also explain the apparent discrepancies between different systems which have been reported and suggests that a 'general' rule regarding the target site cleavage rates for ribozymes may not apply. What can be drawn from our results, however, is that if a U:A is the central bp of the target site (i.e., a U in the substrate and an A in the hybridising position on the ribozyme), a relatively high degree of flexibility is tolerated in the other two positions of the
161 cleavage I00
(a) (b) (c)
0o
(a) (b) (c)
(a) (b) (c)
(a)
(b)(c)
ACC CCC GCC UCC ACC UG +UG CCC GG +GG GCC CG +CG UCC AG +AG
f Com
60
40 20 0 o.oo
.
.
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.
.
~:
l.oo
i.ao
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Time (h) -/3-
GUC/CA TlOX--Omin
-.--t-- CAC/GU TIOE=2h
AAC/UU TlO,~.=4h
~
~
Rz
Sub
LIAC/AO Tl0,~,=l.fh
(;AC/CU No reaction
Fig. 5. A time course involving XAC/X'U substrate-ribozyme interactions. The GUC/CA target site cleavage rate is also illustrated as a control. T.j.,,.. the time required for the reaction to achieve 10% of product formation.
triplet. This is consistent with observations made with single nt changes by Koizumi et al. (1988a).
(e) Site-specific cleavage occurs with changes to the middle residue of the GUC triplet Although a U residue in the central position of the triplet appears necessary for efficient cleavage, we have observed that certain XAC and XCC triplets can also be cleaved. Results in Fig. 5 show that certain XAC substrates, in the presence of the appropriate X'U ribozyme, will cleave specifically, although at a markedly reduced rate. While double mutations AAC/UU, CAC/GU and UAC/AU all exhibited cleavage, the GAC substrate did not cleave. T~/2 values for the three constructs capable of cleaving were not obtained due to the slow rate of reaction. However, T~o% values were 4, 2, and 1.5 h, respectively. The corresponding Tioo. for GUC/CA was less than 5 rain in a series of experiments. Target sites containing an XCC triplet also cleave, but rates were too slow to get any indication of significant activity (Fig. 6). Cleavage was only observable following several hours of incubation of concentrated ribozyme/ substrate mixes (i.e., 1.5 pmol substrate and 2pmol ribozyme in a 5-#1 reaction). Within the XCC mutants, the ACC and the UCC target sites were most efficiently cleaved. These are double mutants from the normal GUC target and have not been studied previously. Their increased rate compared to CCC and GCC target sites may be due to greater flexibility afforded by weaker A:U pairing compared with stronger G:C pairing. This is supported by the observation that those XCC target sites comprised exclusively of G:C pairing were very much slower, if exhibiting cleavage at all, than those containing an A:U pair. The ACC/UG and UCC/AG reactions showed greater activity than CCC/GG and GCC/CG. This was not the case for the XGC/X'C substrate/ribozyme interactions which showed no observ-
5'P 3'P Fig. 6. Autoradiograph showing cleavage reactions involving XCC mutant substrate RNAs mixed with the appropriate mutant ribozyme so that base pairing between the two sequences was maintained, i.e., XCC/X'G. The 5-pl reaction volumes contained 1.5 pmoi of substrate and/or 2 pmol of ribozyme, incubated individually or together for 4 h under standard reaction conditions as described in Fig. 2. For each of the XCC/X'G combinations, the lanes show: a, XCC substrate incubated alone; b, X'G ribozyme incubated alone; e, reaction products for XCC substrate and/ X'G ribozyme incubated together. Cleavage reactions and PAGE were carried out as described in Fig. 2. The RNA species are as follows: Sub, substrate RNA; Rz, ribozyme; 5'P, cleavage product representing the sequence 5' ofthe active site; 3' P, 3' cleavage product. The high-Mr band labelled 'Corn' is most likely to be nondenatured substrate/ribozyme complex.
able site-specific cleavage. It indicates that a G as the central residue in the target-site triplet is not tolerated. Only GGC had been tested in previous work and was found not to cleave (Ruffner et al., 1990).
(f) G:U base pairing is tolerated at the target site Non Watson-Crick alignments (termed wobble base pairing) can occur in nucleic acid hybrids. In particular, a G:U pairing has been found to approximate an A:U pairing in stabilising efficiency (Saenger, 1984). It has structural similarities to Watson-Crick base-pairing and therefore does not disrupt the double helix, causing only a slight 'bulge' in the sugar-phosphate backbone (Saenger, 1984). For this reason G:U mismatches were tested in the target site/ribozyme interaction. It was found that cleavage reactions can occur under these conditions (Table I). Two target sites, GUC and UUC, were chosen because they can be cleaved efficiently when normal base pairing is mainrained. This made the assays of relative rates more accurate and also enabled us to observe the effect of wobble pairings in both hybridising positions. In the case of the GUC target, site-specific cleavage was only observed for the GUC/CA normal and GUC/CG
162 TABLE I Effect of G:U 'wobble' base pairing at the target site on cleavage rates Substrate"' Ribozyme
Relative activity (%) h
GUC •
Substrate"' Ribozyme
Relative activity i o~ / O I~b
UUC
.
CA
66 6;,
100 70
U,
120 21
0 o
X6
0 o
" Two target sites. UUC and GUC were incubated with ribozymeswhich introduced G:U base pairing in either and/or both hybridisingpositions. Dots indicate potential base pairing between the substrate target site and four ribozymes. The substrat¢ sequence is shown at the top of each column with the ribozymes listed underneath. h Relativeactivityrepresents the extent of cleavage after 1 h when GUC/ CA is expressed as 100%. Reaction conditions were as outlined in Fig. 2. wobble combinations. Therefore, in this instance a wobble mismatch was tolerated in the second position of the target site but not in the first. However, upon targeting U U C substrate, only U U C / A A normal and U U C / G A wobble combinations showed cleavage. In this case a wobble mismatch is tolerated in the first position but not in the second. G U C / U G and U U C / G G did not cleave indicating that a wobble mismatch at both positions was not tolerated (Table I). The G U C / C G reaction was slower than the correct GUC/CA pairing although rates were still relatively fast (Table I). In comparison, the U U C / G A pairing is markedly slower than its Watson-Crick counterpart. The Tie,. for U U C / G A was 40 rain, whereas the U U C / A A is < 5 rain. Therefore, it appears that wobble base pairing is permissible in some, but not all, positions in the UUC and G U C target sites studied (Table I). Both of the wobble mismatches which showed cleavage had slower cleavage rates when compared with their Watson-Crick counterparts. The G U C / C G pairing was slower than the correct G U C / C A pairing although rates were still relatively fast. Presumably, as the stability of the hybrid is similar, the effect on cleavage is a result of the slight structural deviation from the standard bp geometry. These results are different to those obtained previously for permissible G:U bp at the target site. Sheldon and Symons (1989) found in their system that a G:U bp is tolerated with the G at the first position of the G U C target triplet (i.e., a G in the substrate and a U in the ribozyme) and not in the second. Once again, it is likely that other components contributing to the overall structure are again affecting the specificity with which these ribozymes can induce cleavage. Regarding the other potential wobble base-pairing situation, the U U C / G A pairing is markedly slower than the normal U U C / A A reaction. Such an interaction has not
been studied previously. Perhaps the absence of relatively strong G:C pairing in the U U C target/ribozyme interaction enhances the effect of the wobble pairing. This could explain why the G U C substrate can tolerate a wobble in the second position but U U C cannot. The bp immediately 5' of the target site is C:G. Perhaps this allows U U C to wobble in the first position but not the second. Thus it seems that U:A pairing can be replaced by a U:G as long as there is a G:C pair immediately to the 5' side of it. However, a G:C pairing could not be replaced by a G:U in either of the two cases examined (Table I).
(g) Conclusions Overall, it now appears that a degree of flexibility can be tolerated in the target site of an RNA substrate in the ribozyme/substrate interactions based on the hammerhead cleavage domains. Even certain wobble base pairings can be accommodated. In all but two cases tested a U as the central residue of the target-site triplet permits efficient sitespecific cleavage. An A or C in this position, with certain base pairings in the first position can also undergo cleavage. These results are summarised in Table II. This work indicates that the general ruling of an N U Y target site (Koizumi et al., 1989) may not hold. Instead, a consensus target rule may not be possible as different target sequences TABLE II Summary of the reaction rates of target sites altered by she specific mutagenesis with base pairing maintained between substrate and ribozyme in all cases Efficient cleavage"
Lower efficiencyh
No activity~
Target Relative Target Relative Target Relative sited activity(°.8)~ site'~ activity(%)~ sited activity(',<.,)~ GUC GUA GUU UUC CUC
100 93 68 120 95
CAC UAC AAC CCC UCC ACC GCC
4 6 3
GAC GUG AUC CGC GGC AGC UGC
NR NR NR NR NR NR NR
" Substratetarget site triplets showingefficientrates of cleavage when the wt target, GUC, is assigned as 100 (i.e., other rates ale expressed relative to GUC). Substrate target site triplets showing low level rates of cleavage when compared to the wt GUC triplet. As the XCC targets listed here, showed cleavage under conditions of high concentration only, they have not been assigned relative rates. ~" Substrate target site triplets showing no discernable cleavage. d Mutant target sites on substrate RNAs. Each target site was combined with a mutant ribozyme such that full base pairing was maintained. Relative activity of each mutant target site after 1 h incubation under standard reaction conditions as outlined in Fig. 2. The wt target GUC = 100, with rates for other target sites expressed relative to this. NR, no reaction.
163 show different cleavage activities according to their own sequence context along with those of the corresponding ribozyme. As structural models of the catalytic domain and cleavage process are developed, these results will need to be accommodated within these observations. This work also provides valuable information for future manipulations involving ribozymes by indicating a number of new and unique target sites as potential cleavage sites for use both in vitro and in vivo.
ACKNOWLEDGEMENTS
The authors wish to thank Lynda Graf for the synthesis ofthe oligos. Support for part of this work was provided under the Generic Technology component of the Australian Government Industry Research and Development Act, 1986.
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