J. Mol. Biol. (1990) 212, 113-125
Effect of Deletions at Structural Domains of Group II Intron bI1 on Self-splicing in Vitro Jiirgen Bach1 and Carlo Schmelzer lnstitut
fiir
und Mikrobiologie der Universit&t Miinchen Ward-sir. la, D-8000 Miinchen 19, FRG
Gene&
Maria-
(Received 14 July
1989; accepted 27 October 1989)
Some group II introns can undergo a protein-independent splicing reaction with the basic reaction pathway similar to nuclear pre-mRNA splicing and the catalytic functions of some of the structural components have been determined. To identify further functional domains, we have generated an ensemble of partial and complete deletions of domains I, II, III and IV of the self-splicing group II intron bI1 from yeast mitochondria and studied their effects on the splicing reaction in vitro. Our results indicate that domains II and IV, which vary considerably in length and structure among group II introns, do not play a direct role in catalysis but mainly help to ensure the proper interaction between upstream and downstream catalytically active structural elements. Deletions of sub-domains of domain I and in and domain III indicate that these elements are involved in 5’ cleavage by hydrolysis a reaction in tram (exon reopening), and that this function can be inhibited without affecting the normal 5’ cleavage by Lransesterification. Yet, we infer that the helical structures affected by the mutational alterations might not contribute to this reaction mode peg se but that changes within local secondary structures perturb the internal conformatjion of the ribozyme. Furthermore, we have designed an abbreviated version of intron bll i with a length of 542 nucleotides, which is still catalytically active.
1. Introduction
The consensus secondary structure model of group JI introns shows a central wheel anld six key base-paired regions (domains I to VI). Experimental evidence is accumulating that shows that specific domains or subdomains of this structure can be associated with particular catalytic activities of the ribozyme. (1) The base-pairing interaction of exonbinding sites (EBSl and EBS2) located in domain I with corresponding sequences at the 3’ end of the 5’ exon (IBSl and IBSB) helps to stabilize the intermediate splicing complex and determines the 5’ splice site (Jacquier & Rosbash, 1986; (Jacquier & Michel, 1987). (2) The interaction of sequences at the 5’ end of the intron with downstream regions of domain I and of domain VI with the conserved branch-point is necessary for 5’ cleavage ‘lby transesterification and formation of branched intron molecules (van der Veen et aE., 1987; Schmelzer & Miiller, 1987; Altura et al., 1989). (3) Domain V is assumed to activate the 5’ junction for cleava,ge in the normal &s-reaction and to align the branch adenosine of domain VI to the 5’ junction (Jarrell et al., 19883). In vitro, this branch-point dependent 5’ cleavage can be bypassed under altered rea,ction conditions, i.e. in the presence of 500 m&t-KCl. The 5’ splice junction is cleaved by hydrolysis, i.e. by H,O or OH- (Jacquier & Rosbash, 1986), rea,ction
The removal of self-splicing group II organelle introns from pre-mRNAs follows the same twostage reaction pathway as for splicing of nuclear pre-mRNAs (Peebles et al., 1986; Schmelzer & Schweyen, 1986; van der Veen et al., 1986). First, precursor is cleaved at the 5’ junction. the RNA Concurrently, the 5’-terminal G residue of the intron is joined qtia a 5’-2’ phosphodiester bond with an A residue near the 3’ end of the intron. Products of this first step are the liberated 5’ exon and the intron lariat covalently joined to the 3’ exon. Second, the intron lariat intermediate is cleaved at the 3’ splice site by the 3’ OH group of the 5’ exon. This cleavage is coupled with ligation of the exons and the concomitant release of the intron lariat. In contrast to the catalytic activity of self-splicing group II introns, which is intrinsic to the intron structure, splicing of nuclear pre-mRNAs depends on the intermolecular interaction of intron sequences with an extensive set of cellular factors including proteins and snRNPst (for a review, see Sharp, 1987). t Abbreviations used: sn, small nuclear; RNP, ribonucleoprotein; EBS, exon-binding intron binding site; nt, nucleotide(s).
site; IBS, 113
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products are excised linear intron and spliced exons. Subsequently, the ligated exons can be reopened at the ligation junction by intron RNA in a transreaction by hydrolysis releasing free 5’ and 3’ exons (Jarrell et al., 1988a). Theoretically, this reopening of ligated exons can result from eleavage of the substrate still bound to the intron and/or after new binding of dissociated ligated exons. For identification of possible, additional functions of other structural domains, we have examined selfsplicing of class II intron bll after introducing several partial or complete deletions in subdomain d2 of domain I, domains II, III and IV. The function of these domains in the autocatalytic reaction of group II introns is discussed on the basis of our findings.
2. Experimental
Procedures
(a) Materials Restriction enzymes, Klenow poiymerase, bacteriophage T4 DNA ligase and polynucleotide kinase were purchased from Boehringer-Mannheim. Reverse transcriptase and bacteriophage T3 RNA polymerase were
from PL Biochemicals. New England
[E~‘S]UTP
was obtained
from
Nuclear.
3. Results preRNA with a length of 1041 nt was transcribed by T3 polymerase of plasmid BS/bIl A + 24. This plasmid was constructed by recloning the insert of pSP6/bIS+ after exonuclease III digestion of 5’ exon sequences into the BlueScribe vector KS (Schmelzer & Schweyen, 1986). The preRNA contains the entire autocatalytic group II intron bI1 (Fig. l), 35 nucleotides of its 5’ exon with IBS2 and IRS1 (11 nt of the plasmid f24 nt of the original 5’ exon) and 238 nt of the 3’ exon. To investigate the function of different domains in the reaetion pathway, we generated several deletions by oligonucleotide mutagenesis, which affected e&her parts or the entire sequences of domains I; II, III and IV. BS/bIlA+ 24 was chosen as the parent plasmid for the introduction of mutations. Furthermore, to get more insight into what structural elements of intron RNA are involved in 5’ cleavage by transesterification and what general role the ionic environment may be playing on catalysis of group II introns, the reactivity of wild-type and mutant transcripts was tested under different buffer conditions. Tncubations were carried out either in buffer C, which promotes 5’ cleavage by hydrolysis and exon reopening, or buffer S (see Experimental Procedures).
(b) Site-directed mutagenesis The Bluescribe ( - ) plasmid BS/bIlA + 24 containing the entire group II intron bI1 was transformed into coli strain CJ236 (dut-, ung-). SingleEscherichia stranded DNA was isolated, mixed with the oligonucleotide and heated to 95°C. After renaturation, synthesis of the 2nd strand was performed with Klenow polymerase and T4 ligase in the presence of deoxynucleotide triphosphates (Kunkel, 1985). After completion of the reaction, the DNA was transformed into strain JM83. Deletions were analyzed by restriction site analysis and confirmed by DNA sequence analysis.
(c) In vitro
transcription
and self-splicing of RNA
Transcripts, uniformly labelled by [3sS]UTP, were synthesized from BlueScribe KS vectors by in vitro transcription with T3 RNA polymerase, after digestion with EcoRI. Transcriptions were carried out in a 20-~1 reaction containing 2 to 5 pg of template DNA, 50 units of the 6 miv-Mgcl,, 40 m&r-Tris . HCl enzyme, (PH 7-5), 10 mM-dithiothreitol, 4 mi%-spermidine, 500 PM of each ribonucleotide triphosphate and 20 @i of [‘%]UTP for 1.5 h at 37°C. Full-length unspliced molecules (preRNA) were separated on 8 M-urea/5% polyacrylamide gels, autoradiographed, gel extracted and purified as described by Frendewey & Keller (1985). In vitro splicing of purified preRNA molecules was incubation buffer C 20 /Ll of performed in 2 KIIM60 mM-MgCl,, (40 miv-Tris HCl (pH 7.5), spermidine, 500 mm-KCI) or buffer S (40 mMTris-SO, (pH 7.5), 60 miv-MgSO,, 2 mivr-spermidine, 500 miv-(NH,),SO,) at 45°C. The reaction was stopped by precipitation with ethanol. The resulting pellet was washed with 70% (v/v) ethanol before being dried under vacuum and dissolved in gel loading buffer. Products were analysed on 5 oh polyacrylamide gels containing 8 M-urea.
(a) Determination of the functions of domain 41 and IV in the group II intron self-splicing reaction Figure 2(a) shows the time-course of self-splicing assays of preRNA of BS/bIlA+24, which was found to self-splice with the same kinetics as a transcript containing the last 98 nt of the original 5’ exon (Schmelzer & Miiller, 1987). Thus, this construct was used as wild-type reference and corresponding preRNA was incubated for varying times in buffer C and buffer S, respectively. Products were identified by comigration with characterized products of the normal self-splicing reaction. Wild-type preRNA reacted very efficiently in both reaction buffers to yield characteristic products; that is, the excised intron lariat, the linear form of the intron, ligated exons and, for buffer C, free 5’ and 3’ exons as a result of a supposed reaction in trans, exon reopening (Jarrell et al., 1988a). In addition, minor amounts of an RNA species corresponding in size to an intermediate product of the reaction, Figure
the intron-3’
2(b)
exon
lariat,
are detectable.
shows the time-course of the selfsplicing reaction of mutant bIlAd2N under identical conditions, i.e. with buffers C and S, respectively. This mutant carries a partial deletion of domain II, leaving a short helical structure with a -NT’ELI site in the hairpin loop (Fig. l(b)). In the presence of KC1 , transcripts remained highly reaetive with almost the same efficiency as the wild-type reference. However, the amount of lariats is reduced and substantial amounts of a product that migrates above the intron lariat, the putative intron-3’ exon lariat intermediate, accumulate. This product is
-
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J. Bach1 and C. Schmelzer
WT
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L-
5’E 3’E ) 3’E *
Fig. 2.
converted into the lariat form during the course of the reaction by subsequent 3’ cleavage. Only minor amounts of ligated exons are detectable and, in addition, products with a size as expected for the linear intron and the linear intron-3’ exon now accumulate. This observation indicates that 5’ cleavage in buffer C occurred by transesterification and by hydrolysis. The ratio of ligated exons to free exons changes in favour of free products, suggesting an enhanced exon-reopening reaction. The array of reaction products changes drastically when bilAd2N preRNA is incubated in buffer S, which shifts the reaction mode almost exclusively transesterification. Virtually no to bands comigrating with ligated exons and linear excised introns appear, and only minor amounts of lariats can be found. Instead, RNA with the expected size of a free 5’ exon and substantial amounts of the intron-3’ exon lariat accumulate. These results show that an obvious consequence of the deletion is that 3’ cleavage is almost completely eliminated, whereas the reduction of the 5’ cleavage rate, as estimated from the ratio of preRNA to intron-3’ exon lariat, is threefold relative to the wild-type. To gain more insight into the possible function of domain II, a second mutation (bilAd2) was designed in such a way as to yield a clean deletion of
the entire domain but leaving the central wheel of the ribozyme intact (Figs l(a) and 3(a)). Incubation of preRNA with buffer S revealed that it is almost completely unreactive. substantial However, amounts of the intermediate intron-3’ exon exclusively in lariat form (and of the free 5’ exon, which is not visible on the autoradiograph) accumulate after incubation of the preRNA in buffer C. This gives an indication that 5’ cleavage via transesterification still occurs quite efficiently, whereas 3’ cleavage is completely eliminated. These results show that complete removal of domain II increases the phenotypic effect of the partial deletion. Ilowever, since it is obvious that most of the domain II elements are dispensable for the autocatalytic process, we conclude that this domain does not have an important, structural function per se in the self-splicing reaction We next analysed the reaction of preRNA of a domain IV mutant in buffers C and S. Mutant bilAd4 has a clean deletion of stem IV and results of incubations up to five hours are shown in Figure 3(b). Comparison of the time-course of both reactions revealed that the reactivity of the preRNA is almost completely eliminated in buffer S. In contrast, the preRNA retains reactivity in buffer 6, although at a much lower level than with wild-type
117
Group II Intron 611 Self-splicing
(b) bllAd2N
S
C M
012345
012345
L- 3’E L* PW lVS*
5’E - 3’E * 3’E *
Figure 2. Time-course of self-splicing reactions of preRNA from bI1 wild-type and a domain 11 mutant. [3sS]UTP-labelled transcripts from EcoRI-cleaved plasmids were incubated for 0, 5, 10, 15, 30 or 60 min (lanes 0 to 5, respectively) at 45°C. Samples were separated on 8 M-Urea/5% polyacrylamide gels. C, Incubation in reaction buffer C respectively. As a size marker, (wit,h 500 mM-ECl). S, Incubation in reaction buffer S (with 500 miv-(NH,),SO,), characterized reaction products of preRNA from wild-type (bIlA+24) were coelectrophoresed (M). (a) Time-course of the self-splicing reaction from BS/bIlA+24 preRNA (wild-type). The autoradiograph shows a product pattern typical for the self-splicing reaction of class II intron bI1 that is the excised intron lariat (L), the linear form of the intron and/or the broken lariat (IVS). the intron-3’ exon lariat int)ermediate (L-3’ E), ligated exons (5’ E-3’ E) and, for buffer C, free 5’ and 3’ exons as the presumed result of an exon-reopening reaction (Jarrell et al., 1988a.). Due to its weak label, the 5’ exon is not’ visible on the autoradiograph. Additional products that migrate between the primary transcript and the intron lariat can be seen on this and subsequent Figures. These RNA species have not been characterized. (lo) incubation of preRNA from mutant BS/bilAd2N with partial deletion of domain II. Incubation in buffers C and S leads to the accumulation of a product with a molecular weight expected for the intron-3’ exon lariat (L-3’ E), indicating a reduced efficiency of 3’ cleavage. The lower part of the autoradiograph is overexposed to visualize the free 5’ exon.
RNA.
Major
products
are
the
free
reopened
exons
and the excised linear intron; only very faint signals for lariats are detectable, suggesting that a clean deletion of domain IV eliminates the branch-point dependent attack at the 5’ junction almost completely, yet some residual 5’ cleavage by hydrolysis can be achieved. Whereas domain II deletions
mainly affect the second step of the self-splicing process, complete removal of helix IV drastically reduces the efficiency of the first step of the reaction. Since the basis for the 5’ cleavage ought to be the close proximity and the precise alignment of domains V and VI with the 5’ junction, it, is conceivable that the structural change introduced by
(a) bllAd2
c M
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2
c
3
5
s M
012345
5’E
3’E * 3’E -
0
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Group II Intron bIl Xel&dicing domain TV deletion could interfere with this interaction, thus suppressing the first (and consequently also the 2nd) step of splicing.
Deletions in domain III and domain I can suppress 5’ cleavage by hydrolysis and exon reopening in the presence of KC1
(b)
We next analysed the self-splicing reaction of transcripts with several deletions in domain III (Fig. l(b)). The base of this domain, a helical structure with a bulging internal loop, is well-conserved among group II introns and its functional importance is illustrated by mutants in this helix (bIlA4) and in this internal bulge loop (M1301), which abolish splicing *in vitro and in vivo (Schmelzer et al., 1981, 1983; Schmelzer & Schweyen, 1986). Incubation of preR1”;A from mutant bIlA4 under the new reaction conditions led to the same results as previously published (Schmelzer & Schweyen, 1986). Except for some faint lariat bands obtained after incubation in buffer C, no reaction products are detectable (Fig. 4(a)), thus reinforcing our conclusion that this conserved region is important for the splicing process. As a first approach to studying which portions of domain III are essential for the splicing reaction, we successively deleted structural components of this domain, starting with peripheral regions of domain III. which do not share common structural elements among class II introns. The results are shown in Figure 4(b). The deletion of two outer helices with long A. U runs (bIlAd3a) yields a structure of bIldomain 111, which bears resemblance to its counterpart in intron aI5c. As expected, under both reaction conditions, the splicing efficiency and the array of reaction products are indistinguishable from wildtype, revealing that these sequences are not critical for the self-splicing reaction (see Fig. 2(a)). This pattern changes dramatically if a more extensive deletion is introduced, leaving only the conserved helix at the base of domain III with the internal loop and a small hairpin loop (bIlAd3). Whereas the self-splicing reaction in t’he presence of buffer S was completely eliminated, some residual splicing could be observed in buffer C (Fig. 4(c)). Besides unspecific products, which can be seen also in the control lane, two major products arise, the excised intron lariat and the ligated exons. Interestingly, products with sizes as expected for free exons are not detectable, indicating the suppression of the supposed rea&ion in trans, the exon reopening by hydrolysis. Experimental evidence has accumulated that domain f of group II introns fulfils at least two importantj funetions for the self-splicing process.
119
First, mutual recognition of exon-binding sites (the EBSl and EBS2 sequence elements) located at a short distance inside domain I and intron binding sites at the 3’ end of the 5’ exon (IRS1 and IBS2) by base-pairing is necessary for activation and recognition of the 5’ splice site. Mutations, which weaken the stability of the EBS/IBS helix, interfere with 5’ cleavage (Jacquier & Michel, 1987). Second, the simultaneous interaction of nucleotides at the 5’ end of the intron with regions of domain I and of domain VI is necessary for form&ion of branched intron molecules. If one of these structural elements is disturbed, 5’ cleavage by transesterification fails to take place; however, it can be bypassed by hydrolysis under appropriate buffer conditions (van der Veen et al., 1987; Schmelzer & Miiller, 1987; Altura et al., 1989). In order to investigate further possible functions of helix I subdomains, we constructeld a mutant (bIlAdlD2) with a clean deletion of a peripheral non-conserved hairpin structure (stem, D2 of domain I) immediately downstream from EBSB, which does not affect the sequence of the exonbinding site. We expected that this mutation should show no (or only a weak) phenotype. Even if the disturbance of the local structure immediately downstream from EBS2 could interfere with the function of EBS2, the effect should not be significant, as it could be shown that the EBSl/IBSl interaction alone is sufficient for the cis reaction in vitro (Jacquier & Michel, 1987; Miiller et al., 1988). As shown in Figure 5, in buffer S, the mutation leads to a tenfold reduction in preRNA reactivity relative to wild-type; however, the array of products closely resembles that of the wilcl-type RNA with a slightly increased concentration of intron-3’ exon lariat. Splicing in buffer S does not go to completion, even after an incubation period of 60 minutes. (After 10 min incubation, the reaction does not proceed further.) This could indicate that, due to the mutation, most of the RNA molecules are prevented from folding into a catalytically active conformation under these assay conditio’ns. With incubation in KCl, the rate of preRn’A turnover, as estimated from the ratio of remaining preRNA to all reaction products, is indistinguishable from wildtype; however, the distribution of products is different. Predominant RNAs are the excised intron in lariat form and ligated exons, only minor amounts of linear excised introns and/or broken lariats can be found. Most strikingly, even after an incubation period of one hour, only a faint signal for a free 3’ exon can be seen. These observations led us to conclude that, in buffer C, the deletion does not interfere with the normal 5’ cleavage in cis by
Figure 3. Time-course of splicing reactions of complete deletions of domain II or domain IV. Assay conditions were the same as described for Fig. 2. Due to the reduction of reactivity, incubation times were extended (0, 15, 30, 60, 120 or 300 min; lanes 1 to 5). Free 5’ exons are not visible on the autoradiograph because of their size (35 nt) and low concentration. Marker (M) as in Fig. 2. (a) Shown is the time-course of the self-splicing reaction of mutant bflAd2 with a complete deletion of helix II, leaving the central wheel of the ribozyme unaffected. (b) Incubation of preRKA of mutant bPlAd4 wish an exact deletion of helix IV.
b I1 A4
c 012345"01
bll
Ad
3 a
C
L- 3’E ) LP* JVS-
3’E *
3
s
4
5
Group II
-
Intron
bI1 Self-splicing
--
121
(c) bll
Ad3
C
S MO12345
012345
Figure 4. Effect of removing different peripheral regions of domain III on the overall reaction and exon reopening. Assay conditions were as described for Pig. 2. RNAs of mutants (a) bIlA4 and (c) bIlAd3 were assayed wit)h prolonged incubation times (0, 30,60, 120 or 300 min; lanes 0 to 5). (a) bIlA4, which carries a deletion of 6 nt from the S$I site of stem III. ‘l’his deletion abolishes the formation of a conserved short helical region in domain III (Schmelzer & Schweyen, 1986). (lo) bIlAd3a, a deletion of 88 nt of peripheral structures of domain III leaving the conserved areas undisturbed. (c) bIlAd3, a deletion of 135 nt of outer heliees of domain III. Only the conserved short hairpin of 32 nt with an internal bulge loop of the domain III base has been left.
transesterification, but has a pronounced suppression of 5’ cleavage by hydrolysis in trans (reopening of ligated exons).
effect on in cis and
(c) A 542 nucleotide version of bll as the swmllest group II intron capable of self-splicing Tn order to synthesize a group II ribozyme with a -minimum length, we constructed a version of bI1 reduced in size by 226 nt to 542 nucleotides. This was accomplished by combining deletion of helix D2 of domain I (bIlAdlDB), partial deletion of domain II (bIIAD2N) and partial deletion of domain III (bIlAd3a). Results of the self-splicing reaction are shown on the autoradiograph in Figure 6. In buffer S, no spliced RNA is produced; the only detectable product is the intron-3’ exon lariat intermediate, which indicates t,hat some residual 5’ cleavage by transesterification is occurring but that 3 cleavage is completely inhibited. This result resem-
bles the phenotype of the single deletion bIld2N and shows, as expected, that a con&nation of several deletions cannot compensate for the effects of single deletions, from which they were constructed. The rate of the self-splicing reaction is largely increased by incubation in buffer C with a similar efficiency to that of wild-type prt:RNA. The pattern of products reflects the phenotype of either of the single deletions; decreased exon reopening (mutant bIlAdlD2) and accumulation of the intron-3’ exon lariat (mutant bIlAd2N).
4. Discussion In this work, we have analysed structural domains I, II, III and IV of group IL intron bIl (subclass IIR; Michel et al., 1989) with respect to their function in the autocatalytic splicing reaction in cis and in trans. This was accomplished by constructing various deletions of (sub)domains of
122
J. Bach1 and C. Xchwdzer bllAdlD2
012345
1
2
3
4
5
-“‘E Pm IVS-
5’E 3’E *
Figure 5. Analysis of the self-splicing products of a helix I subdomain d2 deletion mutant. bIlAdlD2 carries a deietion of 48 nt removing a peripheral non-conserved helix d2 of domain I immediately downstream from exon binding site 2 (dlDX-wt, EBSX). preRNA was spliced in vitro as described for Fig. 2.
the intron and studying their effect on self-splicing in vitro. We showed that removal of most of the nucleotides of domain II, leaving only a short hairpin at its base (mutant bIlAd2N), has no pronounced effect on the splicing efficiency, whereas the complete deletion (mutant bIlAd2) abolishes the reaction almost completely. These findings are in agreement with the observations reported by Kwakman et al. (1989), who could obtain similar effect’s with domain II deletions of the related group II intron aI%. The simplest explanation for this observation is that this domain is functionally irrelevant. It might merely provide a minimum of nucleotides (or structures) to act as a joining element, which links upstream and downstream domains for bringing reactive groups in close proximity and might help to ensure a critical spacing between domain I and downstream catalytical elements; complete deletion could interfere with the interaction of these domains by causing a steric hindrance, leading to a complete loss of reactivity. This hypothesis would be consistent with the findings obtained by comparative computer analysis, which show that within group II introns, domain II lacks any conserved regions and varies in length
from I9 to 244 nucleotides (among subclass IIB; Michel et al., 1989). On the other hand, under reaction conditions that we assume to allow more flexibility of the RNA structure, the ability (at least) for 5’ cleavage can be This can be achieved by leaving a rest.ored. minimum portion of stem II (e.g. the small hairpin of mutant bIlAd2N) or by incubation in KCl, which presumably allows a less rigid structure of the intron and could facilitate the folding into an active conformation, thus leading to a less severe reduction of reactivity. Under these conditions, the wild-type phenotype is not fully restored. The accumulation of substantial amounts of the free 5’ exon and the intron-3’ exon lariat intermediate reveals a stong inhibition of cleavage at the 3’ junction. Obviously, the structural alteration of the ribozyme still allows the correct positioning of both domains V and VI to the 5’ junction, with the subsequent cleavage by attack of the bra.nch 2’ OH group but blocks the second step of the reaction; the 5’ exon cannot attack the 3’ splice site. One speculative interpretation of this effect could be that the proper interaction of domain I with domains III to VI is required for the stabilization of a transition state, e.g. by helping to prevent the 5’ exon and the
Group II Intron bIl Self-splicing
_____~
123
bllSM1
C 012345’
S 012345
L 3’E L, P*
3’E -
Figure 4. Splicing activity of a triple deletion mutant of bI1. For construction of this mutant b&ml, deletion of helix and partial deletion of domain IIT (bIlAd%) were partial deletion of domain II (bIlAd2N) D2 of domain I (bIlAdlDZ), combined. PreRNA was incubated at 45°C for 0, 30, 60, 120 or 300 min (lanes 1 to 5, respectively) in buffer C (C) or buffer S (8)
ribozyme, which are held together by the EBS/IBS pairings, from diffusing away from each other before 3’ cleavage. Deletions of domain II could facilitate a premature release of the 5’ exon from the exonbinding sites before it can attack the 3’ junction. As the 3’ 0II group of the 5’ exon can attack the 3’ splice site only when associated with the intron, this would result in a block of the second step of splicing. Anot,her explanation could be that the deletion hampers a possible conformational change of the ribozyme during transition from the first to the second reaction step, thus freezing the ribozyme in a conformation that cannot perform 3’ cleavage. Domain IV, which also shows only weak structural conservation, separates domain V and domain VI from the rest of the ribozyme. Deletion of domain IV of intron bI1 does not result in complete abolition of self-splicing and we conclude that this helix has a similar function to that of domain II as a connecting link, i.e. its elimination could reduce the flexibility of the ribozyme and could disturb the alignment of upstream ribozyme structures with domains V and VI, which is required for 5’ cleavage. There are additional observations to support the hypothesis that domain IV does not have a catalytic function per se. Experimental evidence has
been offered that two half-molecules of the related intron group II intron aI%, which ase split at domain 4, splice efficiently and accurately in a tram reaction (Jarrell et al., 19886). At first sight, our results of the reaction in cis seem to be in contrast to the observations replorted by Jarrell et al. (1988b), who showed that a deletion of domain IV of intron aI5c did not have a measurable effect on splicing efficiency in the normal cis reaction. However, this could be explained by the fact that, in this study, domain IV was not exactly deleted but replaced by 13 nucleotides o-f the plasmid, which might have led to a less stringent reduction of flexibility of the intron molecules or could fall short of a critical spacing requirement between domains V and VI and upstream catalytic elements. It was first shown by Jacquier & Rosbash (1986) that in a trams-reaction of free 5’ exon-G molecules and the intron, trans.splicing could occur without any branch-point formation, and that the 5’ junction was cleaved by hydrolysis; i.e. the 2’ OH group of the branch as the nucleophilic agent in the normal &s-reaction can be substituted by OH- or water. Jarrell et ~2. (1988a) showed that, under appropriate reaction conditions, 5’ cleavage in cis
124
J. Bach1 and 6. Schmelzer
can occur by hydrolysis; under these conditions, ligated exons can be reopened in a trans reaction. For this reaction in trans, the alignment of the substrate (ligated exons) with the intron requires only the EBS/IBS base-pairing, whereas positioning of the 5’ junction in the reaction in C~S is obtained by the EBS/IBS base-pairing and by the covalent linkage of the 5’ exon with the intron. Here, we show that deletions of subdomains of domains I and III can abolish 5’ cleavage by hydrolysis and exon reopening. Deletion of domain III also affects the normal 5’ cleavage by transesterification; thus, this structural element seems to participate in both cleavage modes. These findings are consistent with previous in viva studies, in which we showed that mutations in the conserved region of domain III (M1301) completely abolish splicing in vivo, which we assume to occur exclusively by transesterification (Schmelzer et al., 1981, 1983). It is worth noting that, in viwo, mutation Ml301 can be suppressed by a nuclear allele-specific suppressor SUP-101. Thus, it is tempting to speculate that a key role of this domain in vivo could be its interaction with nuclear protein components, which could accelerate the rate of the splicing reaction in viva (Schmelzer et al., 1983; Schmidt et al., 1987). Alteration of the local secondary structure of helix I by deletion of a non-conserved hairpin structure (D2) does not interfere with the 5’ cleavage by transesterification but suppresses hydrolysis at the 5’ splice site and exon reopening (reaction in trans). Thus, the result of this deletion of stem d2 of domain I reveals a novel mutant phenotype. It selfsplices with the same kinetics as the wild-type under appropriate reaction conditions; however, unexpectedly, the set of products obtained in the presence of added KC1 is the same as for the wild-type transcript in the (NH,),SO,+-containing reaction. Obviously, in contrast to the wild-type situation, be shifted from the 5’ cleavage mode cannot transesterification to hydrolysis. One interpretation of this result could be that the conformation of the mutant RNA in the presence of KC1 is similar to that of the wild-type in which excludes the nucleophilic attack N%),SO,> of hydroxide ions or water at the 5’ junction and thus impedes the alternative reaction pathway, i.e. 5’ cleavage in cis by hydrolysis. Furthermore, we showed that the exon reopening reaction of this mutant is almost completely eliminated. If exon mechanistically, to a reopening corresponds, 5’ cleavage step by hydrolysis, then this interpretation would be consistent with our assumption that this mutant is incapable of hydrolytic 5’ cleavage (neither in cis nor in trans). On the other hand, we cannot exclude the possibility that the mutational alteration of the ribozyme specifically affects the intermolecular cleavage reaction mode. One reasonable interpretation could be that, after completion of the normal self-splicing reaction, a conformational change of the ribozyme might mediate the rapid dissociation of the substrate-enzyme
complex. Partial deletions of domain I, which do not affect 5’ cleavage in cis by transesterification but have a marked effect for the reaction in trans, could alter the spatial structure of the ribozyme in such a way that exon reopening is prevented either (1) by speeding up the dissociation of the ligated exons from the enzyme and/or impairing new binding of the ligated exons to EBS sequences of the intron, or (2) by permitting binding of the substrate but not its cleavage in trans. As in the case of our interpretation of the e&&s of domains IT and IV deletions, we do not conclude that the deleted portions of domain I and III are important for the hydrolytic or trans-reaction mode also refer to nonper se, since these structures conserved helical regions almost exclusively with long runs of A.U, but that their absence could modify the overall conformation of the ribozyme, leading to the impediment of these reactions. The results obtained by studying the self-splicing reaction of triple mutant bIlSM1 show that of non-conserved distal regions leaving the removal catalytic core of the ribozyme untouched do not necessarily yield ribozymes with the same inetics of reaction as the wild-type, and it is reasonable to assume that the deletions could influence the stability and function of the whole structure. On the other hand, it is conceivable that this mutilated intron requires some changes in the reaction conditions to reveal its optimum activity. Yet, despite these potential limitations, the ereation of an even smaller group II ribozyme by introduction of additional deletions seems to be promising.
We thank Ana Maria Merlos, Francoise Davison-Brunei and Horst Domdey for critical reading of the manuscript, Barbara Geihaus for her expert technical assistance and Diethard Tautz for synthesis of the oligonucleotides. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
References Altura, R., Rymond, B., Seraphin, B. & Rosbash, M. (1989). Nuel. Acids Res. 17, 335-354. Frendewey, D. & Keller, W. (1985). Cell, 42, 355-367. Jacquier, A. & Michel, F. (1987). Cell, 50, 17-29. Jacquier. A. t Rosbash, M. (1986). Science, 234; 1099-I 104. Jarrell, K. A., Peebles, C. L., Dietrich, R., Romiti, S. L. & Perlman, P. 5. (198%). J. Biol. Chem. 263, 3432-3439.
Jarrell, K. A., Dietrich, R. C. & Perlman, P. S. (198%). Cell. Mol. Riol. 8, 2361-2366. Kunkel, T. A. (1985). Yroc. Nat. Acad. b’ci., U.S.A. 82, 488-492. Kwakman, J. H. J. M., Konings, D., Pel, H. J. & Grivel!, L. A. (1989). Nucl. Acids Res. 17; 4205-4215. Michel, F.; Umesono; K. & Oseki, H. (1989). Crene, in the press. Miiller, M. W. M., Schweyen, R. J. & Schmelzer, C. (1988). Nuel. Acids Res. 16, ‘7383-7395.
Group II
Intron
Peeb!es, 6. L., Perlman, P. S., Mecklenburg, K. L., Petrillo, M. L., Tabor, J. H., Jarrell, K. A. & Cheng, H.-L. (1986). Cell, 44, 213-223. Schmidt, C., Sollner, T. & Schweyen, R. J. (1987). Mol. Gen. Genet. 210, 145-152. Schmelzer, 6. & Miiller, M. W. (1987). Cell, 51, 7533762. Schmelzer, C. & Schweyen, R. J. (1986). Cell, 46,557-565. Schmelzer, C., Haid, A,, Grosch, G., Schweyen, R. J. & Kaudewitz, F. (1981). J. Bid. Chem, 256, 7610-7619. Edited
bI1 SelJ”splicing
125
Schmelzer, C., Schmidt, C., May, K. $ Schweyen, R. J. (1983). EMBO J. 2, 2047-2052. Sharp, P. A. (1987). Science, 235, 766-771. van der Veen, R., Arnberg, A. C.. Van der Horst, G., Bonen, L., Tabak, H. F. & Grivell. L. A. (1986). Cell, 44, 225-234. van der Veen, R., Kwakman, J. H. J. M. & Grivell, L. A. (1987). EMBO J. 6. 3827-3831.
by 8. Brenner
Effect of Deletions at Structural Domains of Group II Intron bI1 on Self-splicing in Vitro Jiirgen Bach1 and Carlo Schmelzer lnstitut
fiir
und Mikrobiologie der Universit&t Miinchen Ward-sir. la, D-8000 Miinchen 19, FRG
Gene&
Maria-
(Received 14 July
1989; accepted 27 October 1989)
Some group II introns can undergo a protein-independent splicing reaction with the basic reaction pathway similar to nuclear pre-mRNA splicing and the catalytic functions of some of the structural components have been determined. To identify further functional domains, we have generated an ensemble of partial and complete deletions of domains I, II, III and IV of the self-splicing group II intron bI1 from yeast mitochondria and studied their effects on the splicing reaction in vitro. Our results indicate that domains II and IV, which vary considerably in length and structure among group II introns, do not play a direct role in catalysis but mainly help to ensure the proper interaction between upstream and downstream catalytically active structural elements. Deletions of sub-domains of domain I and in and domain III indicate that these elements are involved in 5’ cleavage by hydrolysis a reaction in tram (exon reopening), and that this function can be inhibited without affecting the normal 5’ cleavage by Lransesterification. Yet, we infer that the helical structures affected by the mutational alterations might not contribute to this reaction mode peg se but that changes within local secondary structures perturb the internal conformatjion of the ribozyme. Furthermore, we have designed an abbreviated version of intron bll i with a length of 542 nucleotides, which is still catalytically active.
1. Introduction
The consensus secondary structure model of group JI introns shows a central wheel anld six key base-paired regions (domains I to VI). Experimental evidence is accumulating that shows that specific domains or subdomains of this structure can be associated with particular catalytic activities of the ribozyme. (1) The base-pairing interaction of exonbinding sites (EBSl and EBS2) located in domain I with corresponding sequences at the 3’ end of the 5’ exon (IBSl and IBSB) helps to stabilize the intermediate splicing complex and determines the 5’ splice site (Jacquier & Rosbash, 1986; (Jacquier & Michel, 1987). (2) The interaction of sequences at the 5’ end of the intron with downstream regions of domain I and of domain VI with the conserved branch-point is necessary for 5’ cleavage ‘lby transesterification and formation of branched intron molecules (van der Veen et aE., 1987; Schmelzer & Miiller, 1987; Altura et al., 1989). (3) Domain V is assumed to activate the 5’ junction for cleava,ge in the normal &s-reaction and to align the branch adenosine of domain VI to the 5’ junction (Jarrell et al., 19883). In vitro, this branch-point dependent 5’ cleavage can be bypassed under altered rea,ction conditions, i.e. in the presence of 500 m&t-KCl. The 5’ splice junction is cleaved by hydrolysis, i.e. by H,O or OH- (Jacquier & Rosbash, 1986), rea,ction
The removal of self-splicing group II organelle introns from pre-mRNAs follows the same twostage reaction pathway as for splicing of nuclear pre-mRNAs (Peebles et al., 1986; Schmelzer & Schweyen, 1986; van der Veen et al., 1986). First, precursor is cleaved at the 5’ junction. the RNA Concurrently, the 5’-terminal G residue of the intron is joined qtia a 5’-2’ phosphodiester bond with an A residue near the 3’ end of the intron. Products of this first step are the liberated 5’ exon and the intron lariat covalently joined to the 3’ exon. Second, the intron lariat intermediate is cleaved at the 3’ splice site by the 3’ OH group of the 5’ exon. This cleavage is coupled with ligation of the exons and the concomitant release of the intron lariat. In contrast to the catalytic activity of self-splicing group II introns, which is intrinsic to the intron structure, splicing of nuclear pre-mRNAs depends on the intermolecular interaction of intron sequences with an extensive set of cellular factors including proteins and snRNPst (for a review, see Sharp, 1987). t Abbreviations used: sn, small nuclear; RNP, ribonucleoprotein; EBS, exon-binding intron binding site; nt, nucleotide(s).
site; IBS, 113
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products are excised linear intron and spliced exons. Subsequently, the ligated exons can be reopened at the ligation junction by intron RNA in a transreaction by hydrolysis releasing free 5’ and 3’ exons (Jarrell et al., 1988a). Theoretically, this reopening of ligated exons can result from eleavage of the substrate still bound to the intron and/or after new binding of dissociated ligated exons. For identification of possible, additional functions of other structural domains, we have examined selfsplicing of class II intron bll after introducing several partial or complete deletions in subdomain d2 of domain I, domains II, III and IV. The function of these domains in the autocatalytic reaction of group II introns is discussed on the basis of our findings.
2. Experimental
Procedures
(a) Materials Restriction enzymes, Klenow poiymerase, bacteriophage T4 DNA ligase and polynucleotide kinase were purchased from Boehringer-Mannheim. Reverse transcriptase and bacteriophage T3 RNA polymerase were
from PL Biochemicals. New England
[E~‘S]UTP
was obtained
from
Nuclear.
3. Results preRNA with a length of 1041 nt was transcribed by T3 polymerase of plasmid BS/bIl A + 24. This plasmid was constructed by recloning the insert of pSP6/bIS+ after exonuclease III digestion of 5’ exon sequences into the BlueScribe vector KS (Schmelzer & Schweyen, 1986). The preRNA contains the entire autocatalytic group II intron bI1 (Fig. l), 35 nucleotides of its 5’ exon with IBS2 and IRS1 (11 nt of the plasmid f24 nt of the original 5’ exon) and 238 nt of the 3’ exon. To investigate the function of different domains in the reaetion pathway, we generated several deletions by oligonucleotide mutagenesis, which affected e&her parts or the entire sequences of domains I; II, III and IV. BS/bIlA+ 24 was chosen as the parent plasmid for the introduction of mutations. Furthermore, to get more insight into what structural elements of intron RNA are involved in 5’ cleavage by transesterification and what general role the ionic environment may be playing on catalysis of group II introns, the reactivity of wild-type and mutant transcripts was tested under different buffer conditions. Tncubations were carried out either in buffer C, which promotes 5’ cleavage by hydrolysis and exon reopening, or buffer S (see Experimental Procedures).
(b) Site-directed mutagenesis The Bluescribe ( - ) plasmid BS/bIlA + 24 containing the entire group II intron bI1 was transformed into coli strain CJ236 (dut-, ung-). SingleEscherichia stranded DNA was isolated, mixed with the oligonucleotide and heated to 95°C. After renaturation, synthesis of the 2nd strand was performed with Klenow polymerase and T4 ligase in the presence of deoxynucleotide triphosphates (Kunkel, 1985). After completion of the reaction, the DNA was transformed into strain JM83. Deletions were analyzed by restriction site analysis and confirmed by DNA sequence analysis.
(c) In vitro
transcription
and self-splicing of RNA
Transcripts, uniformly labelled by [3sS]UTP, were synthesized from BlueScribe KS vectors by in vitro transcription with T3 RNA polymerase, after digestion with EcoRI. Transcriptions were carried out in a 20-~1 reaction containing 2 to 5 pg of template DNA, 50 units of the 6 miv-Mgcl,, 40 m&r-Tris . HCl enzyme, (PH 7-5), 10 mM-dithiothreitol, 4 mi%-spermidine, 500 PM of each ribonucleotide triphosphate and 20 @i of [‘%]UTP for 1.5 h at 37°C. Full-length unspliced molecules (preRNA) were separated on 8 M-urea/5% polyacrylamide gels, autoradiographed, gel extracted and purified as described by Frendewey & Keller (1985). In vitro splicing of purified preRNA molecules was incubation buffer C 20 /Ll of performed in 2 KIIM60 mM-MgCl,, (40 miv-Tris HCl (pH 7.5), spermidine, 500 mm-KCI) or buffer S (40 mMTris-SO, (pH 7.5), 60 miv-MgSO,, 2 mivr-spermidine, 500 miv-(NH,),SO,) at 45°C. The reaction was stopped by precipitation with ethanol. The resulting pellet was washed with 70% (v/v) ethanol before being dried under vacuum and dissolved in gel loading buffer. Products were analysed on 5 oh polyacrylamide gels containing 8 M-urea.
(a) Determination of the functions of domain 41 and IV in the group II intron self-splicing reaction Figure 2(a) shows the time-course of self-splicing assays of preRNA of BS/bIlA+24, which was found to self-splice with the same kinetics as a transcript containing the last 98 nt of the original 5’ exon (Schmelzer & Miiller, 1987). Thus, this construct was used as wild-type reference and corresponding preRNA was incubated for varying times in buffer C and buffer S, respectively. Products were identified by comigration with characterized products of the normal self-splicing reaction. Wild-type preRNA reacted very efficiently in both reaction buffers to yield characteristic products; that is, the excised intron lariat, the linear form of the intron, ligated exons and, for buffer C, free 5’ and 3’ exons as a result of a supposed reaction in trans, exon reopening (Jarrell et al., 1988a). In addition, minor amounts of an RNA species corresponding in size to an intermediate product of the reaction, Figure
the intron-3’
2(b)
exon
lariat,
are detectable.
shows the time-course of the selfsplicing reaction of mutant bIlAd2N under identical conditions, i.e. with buffers C and S, respectively. This mutant carries a partial deletion of domain II, leaving a short helical structure with a -NT’ELI site in the hairpin loop (Fig. l(b)). In the presence of KC1 , transcripts remained highly reaetive with almost the same efficiency as the wild-type reference. However, the amount of lariats is reduced and substantial amounts of a product that migrates above the intron lariat, the putative intron-3’ exon lariat intermediate, accumulate. This product is
-
116
J. Bach1 and C. Schmelzer
WT
C 0123
s M
45
012345
L -3’E,
L-
5’E 3’E ) 3’E *
Fig. 2.
converted into the lariat form during the course of the reaction by subsequent 3’ cleavage. Only minor amounts of ligated exons are detectable and, in addition, products with a size as expected for the linear intron and the linear intron-3’ exon now accumulate. This observation indicates that 5’ cleavage in buffer C occurred by transesterification and by hydrolysis. The ratio of ligated exons to free exons changes in favour of free products, suggesting an enhanced exon-reopening reaction. The array of reaction products changes drastically when bilAd2N preRNA is incubated in buffer S, which shifts the reaction mode almost exclusively transesterification. Virtually no to bands comigrating with ligated exons and linear excised introns appear, and only minor amounts of lariats can be found. Instead, RNA with the expected size of a free 5’ exon and substantial amounts of the intron-3’ exon lariat accumulate. These results show that an obvious consequence of the deletion is that 3’ cleavage is almost completely eliminated, whereas the reduction of the 5’ cleavage rate, as estimated from the ratio of preRNA to intron-3’ exon lariat, is threefold relative to the wild-type. To gain more insight into the possible function of domain II, a second mutation (bilAd2) was designed in such a way as to yield a clean deletion of
the entire domain but leaving the central wheel of the ribozyme intact (Figs l(a) and 3(a)). Incubation of preRNA with buffer S revealed that it is almost completely unreactive. substantial However, amounts of the intermediate intron-3’ exon exclusively in lariat form (and of the free 5’ exon, which is not visible on the autoradiograph) accumulate after incubation of the preRNA in buffer C. This gives an indication that 5’ cleavage via transesterification still occurs quite efficiently, whereas 3’ cleavage is completely eliminated. These results show that complete removal of domain II increases the phenotypic effect of the partial deletion. Ilowever, since it is obvious that most of the domain II elements are dispensable for the autocatalytic process, we conclude that this domain does not have an important, structural function per se in the self-splicing reaction We next analysed the reaction of preRNA of a domain IV mutant in buffers C and S. Mutant bilAd4 has a clean deletion of stem IV and results of incubations up to five hours are shown in Figure 3(b). Comparison of the time-course of both reactions revealed that the reactivity of the preRNA is almost completely eliminated in buffer S. In contrast, the preRNA retains reactivity in buffer 6, although at a much lower level than with wild-type
117
Group II Intron 611 Self-splicing
(b) bllAd2N
S
C M
012345
012345
L- 3’E L* PW lVS*
5’E - 3’E * 3’E *
Figure 2. Time-course of self-splicing reactions of preRNA from bI1 wild-type and a domain 11 mutant. [3sS]UTP-labelled transcripts from EcoRI-cleaved plasmids were incubated for 0, 5, 10, 15, 30 or 60 min (lanes 0 to 5, respectively) at 45°C. Samples were separated on 8 M-Urea/5% polyacrylamide gels. C, Incubation in reaction buffer C respectively. As a size marker, (wit,h 500 mM-ECl). S, Incubation in reaction buffer S (with 500 miv-(NH,),SO,), characterized reaction products of preRNA from wild-type (bIlA+24) were coelectrophoresed (M). (a) Time-course of the self-splicing reaction from BS/bIlA+24 preRNA (wild-type). The autoradiograph shows a product pattern typical for the self-splicing reaction of class II intron bI1 that is the excised intron lariat (L), the linear form of the intron and/or the broken lariat (IVS). the intron-3’ exon lariat int)ermediate (L-3’ E), ligated exons (5’ E-3’ E) and, for buffer C, free 5’ and 3’ exons as the presumed result of an exon-reopening reaction (Jarrell et al., 1988a.). Due to its weak label, the 5’ exon is not’ visible on the autoradiograph. Additional products that migrate between the primary transcript and the intron lariat can be seen on this and subsequent Figures. These RNA species have not been characterized. (lo) incubation of preRNA from mutant BS/bilAd2N with partial deletion of domain II. Incubation in buffers C and S leads to the accumulation of a product with a molecular weight expected for the intron-3’ exon lariat (L-3’ E), indicating a reduced efficiency of 3’ cleavage. The lower part of the autoradiograph is overexposed to visualize the free 5’ exon.
RNA.
Major
products
are
the
free
reopened
exons
and the excised linear intron; only very faint signals for lariats are detectable, suggesting that a clean deletion of domain IV eliminates the branch-point dependent attack at the 5’ junction almost completely, yet some residual 5’ cleavage by hydrolysis can be achieved. Whereas domain II deletions
mainly affect the second step of the self-splicing process, complete removal of helix IV drastically reduces the efficiency of the first step of the reaction. Since the basis for the 5’ cleavage ought to be the close proximity and the precise alignment of domains V and VI with the 5’ junction, it, is conceivable that the structural change introduced by
(a) bllAd2
c M
012345
0
‘9
2
c
3
5
s M
012345
5’E
3’E * 3’E -
0
I
2
4
5
Group II Intron bIl Xel&dicing domain TV deletion could interfere with this interaction, thus suppressing the first (and consequently also the 2nd) step of splicing.
Deletions in domain III and domain I can suppress 5’ cleavage by hydrolysis and exon reopening in the presence of KC1
(b)
We next analysed the self-splicing reaction of transcripts with several deletions in domain III (Fig. l(b)). The base of this domain, a helical structure with a bulging internal loop, is well-conserved among group II introns and its functional importance is illustrated by mutants in this helix (bIlA4) and in this internal bulge loop (M1301), which abolish splicing *in vitro and in vivo (Schmelzer et al., 1981, 1983; Schmelzer & Schweyen, 1986). Incubation of preR1”;A from mutant bIlA4 under the new reaction conditions led to the same results as previously published (Schmelzer & Schweyen, 1986). Except for some faint lariat bands obtained after incubation in buffer C, no reaction products are detectable (Fig. 4(a)), thus reinforcing our conclusion that this conserved region is important for the splicing process. As a first approach to studying which portions of domain III are essential for the splicing reaction, we successively deleted structural components of this domain, starting with peripheral regions of domain III. which do not share common structural elements among class II introns. The results are shown in Figure 4(b). The deletion of two outer helices with long A. U runs (bIlAd3a) yields a structure of bIldomain 111, which bears resemblance to its counterpart in intron aI5c. As expected, under both reaction conditions, the splicing efficiency and the array of reaction products are indistinguishable from wildtype, revealing that these sequences are not critical for the self-splicing reaction (see Fig. 2(a)). This pattern changes dramatically if a more extensive deletion is introduced, leaving only the conserved helix at the base of domain III with the internal loop and a small hairpin loop (bIlAd3). Whereas the self-splicing reaction in t’he presence of buffer S was completely eliminated, some residual splicing could be observed in buffer C (Fig. 4(c)). Besides unspecific products, which can be seen also in the control lane, two major products arise, the excised intron lariat and the ligated exons. Interestingly, products with sizes as expected for free exons are not detectable, indicating the suppression of the supposed rea&ion in trans, the exon reopening by hydrolysis. Experimental evidence has accumulated that domain f of group II introns fulfils at least two importantj funetions for the self-splicing process.
119
First, mutual recognition of exon-binding sites (the EBSl and EBS2 sequence elements) located at a short distance inside domain I and intron binding sites at the 3’ end of the 5’ exon (IRS1 and IBS2) by base-pairing is necessary for activation and recognition of the 5’ splice site. Mutations, which weaken the stability of the EBS/IBS helix, interfere with 5’ cleavage (Jacquier & Michel, 1987). Second, the simultaneous interaction of nucleotides at the 5’ end of the intron with regions of domain I and of domain VI is necessary for form&ion of branched intron molecules. If one of these structural elements is disturbed, 5’ cleavage by transesterification fails to take place; however, it can be bypassed by hydrolysis under appropriate buffer conditions (van der Veen et al., 1987; Schmelzer & Miiller, 1987; Altura et al., 1989). In order to investigate further possible functions of helix I subdomains, we constructeld a mutant (bIlAdlD2) with a clean deletion of a peripheral non-conserved hairpin structure (stem, D2 of domain I) immediately downstream from EBSB, which does not affect the sequence of the exonbinding site. We expected that this mutation should show no (or only a weak) phenotype. Even if the disturbance of the local structure immediately downstream from EBS2 could interfere with the function of EBS2, the effect should not be significant, as it could be shown that the EBSl/IBSl interaction alone is sufficient for the cis reaction in vitro (Jacquier & Michel, 1987; Miiller et al., 1988). As shown in Figure 5, in buffer S, the mutation leads to a tenfold reduction in preRNA reactivity relative to wild-type; however, the array of products closely resembles that of the wilcl-type RNA with a slightly increased concentration of intron-3’ exon lariat. Splicing in buffer S does not go to completion, even after an incubation period of 60 minutes. (After 10 min incubation, the reaction does not proceed further.) This could indicate that, due to the mutation, most of the RNA molecules are prevented from folding into a catalytically active conformation under these assay conditio’ns. With incubation in KCl, the rate of preRn’A turnover, as estimated from the ratio of remaining preRNA to all reaction products, is indistinguishable from wildtype; however, the distribution of products is different. Predominant RNAs are the excised intron in lariat form and ligated exons, only minor amounts of linear excised introns and/or broken lariats can be found. Most strikingly, even after an incubation period of one hour, only a faint signal for a free 3’ exon can be seen. These observations led us to conclude that, in buffer C, the deletion does not interfere with the normal 5’ cleavage in cis by
Figure 3. Time-course of splicing reactions of complete deletions of domain II or domain IV. Assay conditions were the same as described for Fig. 2. Due to the reduction of reactivity, incubation times were extended (0, 15, 30, 60, 120 or 300 min; lanes 1 to 5). Free 5’ exons are not visible on the autoradiograph because of their size (35 nt) and low concentration. Marker (M) as in Fig. 2. (a) Shown is the time-course of the self-splicing reaction of mutant bflAd2 with a complete deletion of helix II, leaving the central wheel of the ribozyme unaffected. (b) Incubation of preRKA of mutant bPlAd4 wish an exact deletion of helix IV.
b I1 A4
c 012345"01
bll
Ad
3 a
C
L- 3’E ) LP* JVS-
3’E *
3
s
4
5
Group II
-
Intron
bI1 Self-splicing
--
121
(c) bll
Ad3
C
S MO12345
012345
Figure 4. Effect of removing different peripheral regions of domain III on the overall reaction and exon reopening. Assay conditions were as described for Pig. 2. RNAs of mutants (a) bIlA4 and (c) bIlAd3 were assayed wit)h prolonged incubation times (0, 30,60, 120 or 300 min; lanes 0 to 5). (a) bIlA4, which carries a deletion of 6 nt from the S$I site of stem III. ‘l’his deletion abolishes the formation of a conserved short helical region in domain III (Schmelzer & Schweyen, 1986). (lo) bIlAd3a, a deletion of 88 nt of peripheral structures of domain III leaving the conserved areas undisturbed. (c) bIlAd3, a deletion of 135 nt of outer heliees of domain III. Only the conserved short hairpin of 32 nt with an internal bulge loop of the domain III base has been left.
transesterification, but has a pronounced suppression of 5’ cleavage by hydrolysis in trans (reopening of ligated exons).
effect on in cis and
(c) A 542 nucleotide version of bll as the swmllest group II intron capable of self-splicing Tn order to synthesize a group II ribozyme with a -minimum length, we constructed a version of bI1 reduced in size by 226 nt to 542 nucleotides. This was accomplished by combining deletion of helix D2 of domain I (bIlAdlDB), partial deletion of domain II (bIIAD2N) and partial deletion of domain III (bIlAd3a). Results of the self-splicing reaction are shown on the autoradiograph in Figure 6. In buffer S, no spliced RNA is produced; the only detectable product is the intron-3’ exon lariat intermediate, which indicates t,hat some residual 5’ cleavage by transesterification is occurring but that 3 cleavage is completely inhibited. This result resem-
bles the phenotype of the single deletion bIld2N and shows, as expected, that a con&nation of several deletions cannot compensate for the effects of single deletions, from which they were constructed. The rate of the self-splicing reaction is largely increased by incubation in buffer C with a similar efficiency to that of wild-type prt:RNA. The pattern of products reflects the phenotype of either of the single deletions; decreased exon reopening (mutant bIlAdlD2) and accumulation of the intron-3’ exon lariat (mutant bIlAd2N).
4. Discussion In this work, we have analysed structural domains I, II, III and IV of group IL intron bIl (subclass IIR; Michel et al., 1989) with respect to their function in the autocatalytic splicing reaction in cis and in trans. This was accomplished by constructing various deletions of (sub)domains of
122
J. Bach1 and C. Xchwdzer bllAdlD2
012345
1
2
3
4
5
-“‘E Pm IVS-
5’E 3’E *
Figure 5. Analysis of the self-splicing products of a helix I subdomain d2 deletion mutant. bIlAdlD2 carries a deietion of 48 nt removing a peripheral non-conserved helix d2 of domain I immediately downstream from exon binding site 2 (dlDX-wt, EBSX). preRNA was spliced in vitro as described for Fig. 2.
the intron and studying their effect on self-splicing in vitro. We showed that removal of most of the nucleotides of domain II, leaving only a short hairpin at its base (mutant bIlAd2N), has no pronounced effect on the splicing efficiency, whereas the complete deletion (mutant bIlAd2) abolishes the reaction almost completely. These findings are in agreement with the observations reported by Kwakman et al. (1989), who could obtain similar effect’s with domain II deletions of the related group II intron aI%. The simplest explanation for this observation is that this domain is functionally irrelevant. It might merely provide a minimum of nucleotides (or structures) to act as a joining element, which links upstream and downstream domains for bringing reactive groups in close proximity and might help to ensure a critical spacing between domain I and downstream catalytical elements; complete deletion could interfere with the interaction of these domains by causing a steric hindrance, leading to a complete loss of reactivity. This hypothesis would be consistent with the findings obtained by comparative computer analysis, which show that within group II introns, domain II lacks any conserved regions and varies in length
from I9 to 244 nucleotides (among subclass IIB; Michel et al., 1989). On the other hand, under reaction conditions that we assume to allow more flexibility of the RNA structure, the ability (at least) for 5’ cleavage can be This can be achieved by leaving a rest.ored. minimum portion of stem II (e.g. the small hairpin of mutant bIlAd2N) or by incubation in KCl, which presumably allows a less rigid structure of the intron and could facilitate the folding into an active conformation, thus leading to a less severe reduction of reactivity. Under these conditions, the wild-type phenotype is not fully restored. The accumulation of substantial amounts of the free 5’ exon and the intron-3’ exon lariat intermediate reveals a stong inhibition of cleavage at the 3’ junction. Obviously, the structural alteration of the ribozyme still allows the correct positioning of both domains V and VI to the 5’ junction, with the subsequent cleavage by attack of the bra.nch 2’ OH group but blocks the second step of the reaction; the 5’ exon cannot attack the 3’ splice site. One speculative interpretation of this effect could be that the proper interaction of domain I with domains III to VI is required for the stabilization of a transition state, e.g. by helping to prevent the 5’ exon and the
Group II Intron bIl Self-splicing
_____~
123
bllSM1
C 012345’
S 012345
L 3’E L, P*
3’E -
Figure 4. Splicing activity of a triple deletion mutant of bI1. For construction of this mutant b&ml, deletion of helix and partial deletion of domain IIT (bIlAd%) were partial deletion of domain II (bIlAd2N) D2 of domain I (bIlAdlDZ), combined. PreRNA was incubated at 45°C for 0, 30, 60, 120 or 300 min (lanes 1 to 5, respectively) in buffer C (C) or buffer S (8)
ribozyme, which are held together by the EBS/IBS pairings, from diffusing away from each other before 3’ cleavage. Deletions of domain II could facilitate a premature release of the 5’ exon from the exonbinding sites before it can attack the 3’ junction. As the 3’ 0II group of the 5’ exon can attack the 3’ splice site only when associated with the intron, this would result in a block of the second step of splicing. Anot,her explanation could be that the deletion hampers a possible conformational change of the ribozyme during transition from the first to the second reaction step, thus freezing the ribozyme in a conformation that cannot perform 3’ cleavage. Domain IV, which also shows only weak structural conservation, separates domain V and domain VI from the rest of the ribozyme. Deletion of domain IV of intron bI1 does not result in complete abolition of self-splicing and we conclude that this helix has a similar function to that of domain II as a connecting link, i.e. its elimination could reduce the flexibility of the ribozyme and could disturb the alignment of upstream ribozyme structures with domains V and VI, which is required for 5’ cleavage. There are additional observations to support the hypothesis that domain IV does not have a catalytic function per se. Experimental evidence has
been offered that two half-molecules of the related intron group II intron aI%, which ase split at domain 4, splice efficiently and accurately in a tram reaction (Jarrell et al., 19886). At first sight, our results of the reaction in cis seem to be in contrast to the observations replorted by Jarrell et al. (1988b), who showed that a deletion of domain IV of intron aI5c did not have a measurable effect on splicing efficiency in the normal cis reaction. However, this could be explained by the fact that, in this study, domain IV was not exactly deleted but replaced by 13 nucleotides o-f the plasmid, which might have led to a less stringent reduction of flexibility of the intron molecules or could fall short of a critical spacing requirement between domains V and VI and upstream catalytic elements. It was first shown by Jacquier & Rosbash (1986) that in a trams-reaction of free 5’ exon-G molecules and the intron, trans.splicing could occur without any branch-point formation, and that the 5’ junction was cleaved by hydrolysis; i.e. the 2’ OH group of the branch as the nucleophilic agent in the normal &s-reaction can be substituted by OH- or water. Jarrell et ~2. (1988a) showed that, under appropriate reaction conditions, 5’ cleavage in cis
124
J. Bach1 and 6. Schmelzer
can occur by hydrolysis; under these conditions, ligated exons can be reopened in a trans reaction. For this reaction in trans, the alignment of the substrate (ligated exons) with the intron requires only the EBS/IBS base-pairing, whereas positioning of the 5’ junction in the reaction in C~S is obtained by the EBS/IBS base-pairing and by the covalent linkage of the 5’ exon with the intron. Here, we show that deletions of subdomains of domains I and III can abolish 5’ cleavage by hydrolysis and exon reopening. Deletion of domain III also affects the normal 5’ cleavage by transesterification; thus, this structural element seems to participate in both cleavage modes. These findings are consistent with previous in viva studies, in which we showed that mutations in the conserved region of domain III (M1301) completely abolish splicing in vivo, which we assume to occur exclusively by transesterification (Schmelzer et al., 1981, 1983). It is worth noting that, in viwo, mutation Ml301 can be suppressed by a nuclear allele-specific suppressor SUP-101. Thus, it is tempting to speculate that a key role of this domain in vivo could be its interaction with nuclear protein components, which could accelerate the rate of the splicing reaction in viva (Schmelzer et al., 1983; Schmidt et al., 1987). Alteration of the local secondary structure of helix I by deletion of a non-conserved hairpin structure (D2) does not interfere with the 5’ cleavage by transesterification but suppresses hydrolysis at the 5’ splice site and exon reopening (reaction in trans). Thus, the result of this deletion of stem d2 of domain I reveals a novel mutant phenotype. It selfsplices with the same kinetics as the wild-type under appropriate reaction conditions; however, unexpectedly, the set of products obtained in the presence of added KC1 is the same as for the wild-type transcript in the (NH,),SO,+-containing reaction. Obviously, in contrast to the wild-type situation, be shifted from the 5’ cleavage mode cannot transesterification to hydrolysis. One interpretation of this result could be that the conformation of the mutant RNA in the presence of KC1 is similar to that of the wild-type in which excludes the nucleophilic attack N%),SO,> of hydroxide ions or water at the 5’ junction and thus impedes the alternative reaction pathway, i.e. 5’ cleavage in cis by hydrolysis. Furthermore, we showed that the exon reopening reaction of this mutant is almost completely eliminated. If exon mechanistically, to a reopening corresponds, 5’ cleavage step by hydrolysis, then this interpretation would be consistent with our assumption that this mutant is incapable of hydrolytic 5’ cleavage (neither in cis nor in trans). On the other hand, we cannot exclude the possibility that the mutational alteration of the ribozyme specifically affects the intermolecular cleavage reaction mode. One reasonable interpretation could be that, after completion of the normal self-splicing reaction, a conformational change of the ribozyme might mediate the rapid dissociation of the substrate-enzyme
complex. Partial deletions of domain I, which do not affect 5’ cleavage in cis by transesterification but have a marked effect for the reaction in trans, could alter the spatial structure of the ribozyme in such a way that exon reopening is prevented either (1) by speeding up the dissociation of the ligated exons from the enzyme and/or impairing new binding of the ligated exons to EBS sequences of the intron, or (2) by permitting binding of the substrate but not its cleavage in trans. As in the case of our interpretation of the e&&s of domains IT and IV deletions, we do not conclude that the deleted portions of domain I and III are important for the hydrolytic or trans-reaction mode also refer to nonper se, since these structures conserved helical regions almost exclusively with long runs of A.U, but that their absence could modify the overall conformation of the ribozyme, leading to the impediment of these reactions. The results obtained by studying the self-splicing reaction of triple mutant bIlSM1 show that of non-conserved distal regions leaving the removal catalytic core of the ribozyme untouched do not necessarily yield ribozymes with the same inetics of reaction as the wild-type, and it is reasonable to assume that the deletions could influence the stability and function of the whole structure. On the other hand, it is conceivable that this mutilated intron requires some changes in the reaction conditions to reveal its optimum activity. Yet, despite these potential limitations, the ereation of an even smaller group II ribozyme by introduction of additional deletions seems to be promising.
We thank Ana Maria Merlos, Francoise Davison-Brunei and Horst Domdey for critical reading of the manuscript, Barbara Geihaus for her expert technical assistance and Diethard Tautz for synthesis of the oligonucleotides. This work was supported by a grant from the Deutsche Forschungsgemeinschaft.
References Altura, R., Rymond, B., Seraphin, B. & Rosbash, M. (1989). Nuel. Acids Res. 17, 335-354. Frendewey, D. & Keller, W. (1985). Cell, 42, 355-367. Jacquier, A. & Michel, F. (1987). Cell, 50, 17-29. Jacquier. A. t Rosbash, M. (1986). Science, 234; 1099-I 104. Jarrell, K. A., Peebles, C. L., Dietrich, R., Romiti, S. L. & Perlman, P. 5. (198%). J. Biol. Chem. 263, 3432-3439.
Jarrell, K. A., Dietrich, R. C. & Perlman, P. S. (198%). Cell. Mol. Riol. 8, 2361-2366. Kunkel, T. A. (1985). Yroc. Nat. Acad. b’ci., U.S.A. 82, 488-492. Kwakman, J. H. J. M., Konings, D., Pel, H. J. & Grivel!, L. A. (1989). Nucl. Acids Res. 17; 4205-4215. Michel, F.; Umesono; K. & Oseki, H. (1989). Crene, in the press. Miiller, M. W. M., Schweyen, R. J. & Schmelzer, C. (1988). Nuel. Acids Res. 16, ‘7383-7395.
Group II
Intron
Peeb!es, 6. L., Perlman, P. S., Mecklenburg, K. L., Petrillo, M. L., Tabor, J. H., Jarrell, K. A. & Cheng, H.-L. (1986). Cell, 44, 213-223. Schmidt, C., Sollner, T. & Schweyen, R. J. (1987). Mol. Gen. Genet. 210, 145-152. Schmelzer, 6. & Miiller, M. W. (1987). Cell, 51, 7533762. Schmelzer, C. & Schweyen, R. J. (1986). Cell, 46,557-565. Schmelzer, C., Haid, A,, Grosch, G., Schweyen, R. J. & Kaudewitz, F. (1981). J. Bid. Chem, 256, 7610-7619. Edited
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Schmelzer, C., Schmidt, C., May, K. $ Schweyen, R. J. (1983). EMBO J. 2, 2047-2052. Sharp, P. A. (1987). Science, 235, 766-771. van der Veen, R., Arnberg, A. C.. Van der Horst, G., Bonen, L., Tabak, H. F. & Grivell. L. A. (1986). Cell, 44, 225-234. van der Veen, R., Kwakman, J. H. J. M. & Grivell, L. A. (1987). EMBO J. 6. 3827-3831.
by 8. Brenner
