Nucleic Acids Research, Vol. 20, No. 15 3897-3904

Splice site selection by intron a13 Saccharomyces cerevisiae

of

the COX1 gene from

Arend J.Winter+, Marian J.A.Groot Koerkamp and Henk F.Tabak* EC Slater Institute for Biochemical Research, Academisch Medisch Centrum, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands Received May 27, 1992; Accepted July 9, 1992

ABSTRACT Interactions of the 5' and 3' splice sites with intron internal sequences of the yeast mitochondrial group I intron a13 were studied using mutation analysis. The results can be fully explained by the splice guide model in which the splice sites are defined by the Internal Guide Sequence. No evidence was found for an alternative interaction between intron nucleotides preceding the 3' splice site and nucleotides in the vicinity of the core region as was found for the Tetrahymena intron. Our results also suggest that binding of the 5' and 3' splice site nucleotides to the IGS can not take place simultaneously. The intron must therefore undergo conformational changes as the reaction proceeds from the first step of self splicing, GTP attack at the 5' splice site, to exon ligation, the second step. INTRODUCTION Introns in precursor RNA transcribed from split genes can be removed in various ways. For example, so called group I and II introns mediate their own excision in a self-splicing process (1, 2, 3). Analysis of the structure of these introns has provided clues to the mechanism of self-splicing, especially with regard to the positioning of the splice sites in the reactive centre. For group I introns this was based on a phylogenetic comparison which showed that the exon sequences flanking the 5' and 3' splice sites are complementary to an intron-internal sequence element called the Internal Guide Sequence (IGS) (4, 5). In the splice guide model of Davies et al. basepairing interactions are proposed between the IGS and the 5' and 3' exon sequences in such a way that the splice sites are always placed close to a conserved guanosine nucleotide within the IGS. This arrangement is invariable between group I introns (4, 6, 7). Since 1982 when the splice guide model was first described, many experiments, involving different group I introns, have been carried out to test it (6, 8, 9, 10, 11). The so called P1 interaction between the 5' exon and the IGS has been demonstrated to exist To whom correspondence should be addressed + Present address: Institut de Biologie Moldculaire et

in several cases (6, 8, 9, 12, 13, 14) (For basepairing nomenclature, see Burke et al. (15)). Definite proof for its existence in the Tetrahymena large rRNA intron was obtained by testing substitution mutants in which basepairing interactions between the 5' exon and IGS are destroyed. Such mutants are less active or defective in 5' splice site recognition and opening, depending on the severity of the substitutions. When double mutants are tested in which compensatory mutations restore basepairing, splicing usually proceeds as normal although there may be some change in reaction rate (9, 16). For the 3' splice site however, the first results obtained with the Tetrahymena intron were somewhat at odds with the splice guide model since destruction of the PIO interaction between the 3' exon and the IGS had no influence on the selection of the 3' splice site and only slightly affected the reaction rate (13). A more detailed phylogenetic comparison using a larger number of group I introns revealed another possible interaction for the selection of the 3' splice site (17). In a significant number of introns including the Tetrahymena intron a basepairing interaction is possible between the two nucleotides immediately downstream of conserved sequence element S and the two nucleotides immediately preceding the intron terminal guanosine residue. This interaction called P9.0 has the potential to bring the 3' splice site in the active centre of the intron in such a way that its position is the same for all introns. Rigourous testing with the Tetrahymena intron has shown that this interaction exists together with the originally proposed PlO pairing and increases the efficiency with which the 3' splice site is selected (18, 19). There are however a number of introns in which the P9.0 interaction consists of only one basepair or does not exist at all. For such introns, the P10 pairing may be the main determinant involved in the selection of the 3' splice site. We tested this possibility for intron a13 of the COXI gene from Saccharomyces cerevisiae. In this intron only one basepairing interaction is possible in P9.0 while there is a potential PIO interaction of four basepairs. Another interesting question which can be addressed with this intron is the importance of the first nucleotides of the intron in the selection of the 5' splice site. In a large number of group I introns this part of the intron can basepair with the IGS at the

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Cellulaire du CNRS,

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Rend Descartes, Strasbourg,

France

3898 Nucleic Acids Research, Vol. 20, No. 15 same position as the 3' exon. This suggests that basepairing between the 3' splice site and the IGS is only possible after cleavage of the 5' splice site and subsequent removal of the 5' intron part from the IGS. We present evidence that this is indeed the case for intron a13.

MATERIALS AND METHODS Construction of a13 mutants Clone pSP64/aI3 containing the wild type a13 intron was digested with Bst EII and Bam HI which both cut unique restriction sites (fig. LA). The two fragments resulting from this digestion were isolated from an agarose gel and the 1423 nt Bst EII -Bam HI fragment was subsequently cut with Mae III. The 337 nt Mae III -Bam HI fragment resulting from this digestion was isolated and ligated in the large fragment obtained from the first digestion which is made possible by the fact that Mae III and Bst ElI produce the same cohesive ends. This results in a 1085 nt deletion of the upstream intron part as compared to the pSP64 a13 construct. Cloning of the deletion version of the aI3 intron behind a T7 promoter was achieved as follows: pSP64 a13/A1085 was digested with Hind III and Hinf I and a 714 nt fragment containing the aI3/A1085 intron plus flanking exons was isolated. The 714 nt fragment was ligated in a Hind III - Sma I digested and phosphatase treated pEP 40 vector in two steps. In the first step the Hind III protruding ends were ligated. In the second step the Hinf I end of the resulting linear molecule was made blunt by filling it in using nucleotide triphosphates and Klenow enzyme. This was followed by a ligation to the Shia I end of the molecule. The resulting construct pT7 a13 (429)/wt was checked by sequence analysis and found to be correct. Construct pT7 a13 (429)/GGGU (fig. IA) was made as follows: pT7 aI3 (429)/wt was digested with Hind III and treated with nuclease BAL 31 to reduce the size of the 5' exon. After digestion with Eco RI, fragments with a length approximately corresponding to the intron plus 3' exon were isolated from a non-denaturing PAA gel and cloned in a Stu I - Eco RI digested pAR 2463 vector. This vector contains a unique Stu I site immediately downstream of the T7 promoter and can be used to keep the number of nucleotides between the T7 promoter and the insert to a minimum of only two basepairs (20). Construct pT7 a13 (429)/GGGU containing a total number of 4 nucleotides of 5' exon was selected from the BAL 31 bank and used in our experiments. Mutant constructs at the IGS and 3' exon in pT7 a13 (429)/wt and pT7 aI3 (429)/GGGU were made by site directed mutagenesis using an M13 derivative of the a13/A1085 construct and appropriate oligonucleotides. Mutations were checked by sequence analysis and fragments containing mutations were cloned in pT7 aI3 (429)/wt and pT7 a13 (429)/GGGU where they replaced the unmutated sequences.

Chemicals and enzymes [a32P]-GTP (400 Ci/mmol) and [a32P]-UTP (3000 Ci/mmol) were from New England Nuclear. Restriction enzymes, ligase and Klenow enzyme were from BRL. AMV reverse transcriptase was from Life Science. RNasin nuclease inhibitor was from Promega. T7 RNA polymerase was home made, using an overproducing E.coli strain kindly provided by F.W.Studier, according to the protocol from Davanloo et al. (20).

Transcription and self-splicing reactions RNA was synthesized using home made T7 RNA polymerase in the presence of 5 units RNasin. Self-splicing was carried out at 40°C in a 20 tl reaction mixture containing 0.2 mM GTP; 100 mM NaCl; 20 mM MgCl2 and 50 mM Tris-HCl pH 7.5. GTP end labelled molecules were formed by incubating unlabelled precursor RNA with 30-40 yCi [a32P]-GTP under the same conditions. The reaction was stopped by adding EDTA to a final concentration of 75 mM in a total volume of 100 ,ul. The products of self-splicing were recovered by passing this mixture through a sephadex G-50 column and precipitating the RNA with ethanol.

RESULTS The deletion mutant of intron a13 reacts like a normal group I intron The self-splicing reaction of the wild type a13 intron from the COX1 gene of Saccharomyces cerevisiae has been studied in detail before (21). Apart from carrying out the normal reaction, this intron is involved in several side reactions of which the formation of interlocked RNA circles is the most striking one. The formation of these circles involves an intron-internal opening site and is explained by making alternative use of the IGS. In order to avoid such side reactions, the experiments described in this paper were carried out with a shortened form of intron aI3 which only carries the specific group I conserved sequence elements. The internal opening site of the wild type construct divides the intron in two parts of 1083 and 431 nts (fig. IA, pSP64 a13). The 5' part contains a large open reading frame. The 3' part contains all of the conserved elements implicated in self-splicing (indicated with IGS, P, Q, R and S). The 5' part of the intron, including two nucleotides of the downstream part, was deleted resulting in a construct in which the first three nucleotides of the intron are unchanged. This results in a 429 nt intron with wild type splice sites (fig. lA and B, pT7 a13 (429)/wt). RNA transcribed from this construct is still capable of performing a self-splicing reaction (fig. 2A). Length measurement and migration behaviour after separation by PAA gel electrophoresis show that the splicing pattern is very simple compared to that of wild type intron: transesterifications take place at the splice sites leading to the formation of ligated exons and excised intron as indicated by the cartoons in figure 2 (panel A, lane R). The excised intron circularizes to an intron of 429 nts which migrates to a position above that of the precursor RNA. Intermediates of the reaction and side products also occur: cleavage products of GTP attack at the 5' splice site and hydrolysis at the 3' splice site. The intron and intron-3' exon products that have a 32p labelled guanosine nucleotide covalently attached to their 5' termini are visible in lane G of figure 2 (panel A). An IGS mutation activates a cryptic 5' splice site and a mutation in the 3' exon abolishes 3' splice site recognition The splice guide model predicts that at the start of the self-splicing reaction the 5' exon is basepaired to the IGS. In many introns, including intron aI3 this interaction also involves intron nucleotides. This makes the interaction more stable (fig. 1 B), which would exclude simultaneous binding of the 3' exon to the IGS via conventional basepairing.

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Nucleic Acids Research, Vol. 20, No. 15 3899

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UGCCGU$KXAUAAUU t11A A A A UG li_i I iii.llli l ISIIIIIhIIi'i Figure 1. aI3 constructs. (A) pT7 aI3 (429)/wt was constructed from pSP64 aI3 by deletion of a 1085 nt intron fragment and subsequent cloning of the deleted intron version behind a T7 promoter. The 3' exon size was reduced as a consequence of the cloning procedure. pT7 aI3 (429)/GGGU was derived from pT7 a13 (429)/wt by shortening the 5' exon. IGS: Internal Guide Sequence, P; Q; R and S: conserved sequence elements; 10: internal opening site. Black boxes: yeast exon sequences; open box: intron internal open reading frame; thick lines: yeast intron sequences; thin lines: vector sequences; flag: T7 promoter (pT7 constructs) or SP6 promoter (pSP 64 construct); Restriction sites: B: Bam HI, BE: Bst EII, E: Eco RI H: Hind III, Hf: Hinf I, M: Mae mI. (B) Alignments of the exon nucleotides with the IGS of the constructs shown in panel A. P1 and PIO are indicated with solid lines. the extension of P1 into the intron is indicated with a dashed line. Shaded nucleotides: exon sequences; outlined nucleotides: IGS; most likely basepairings are shown with solid lines, alternative pairings with dashed lines.

Because the 5' exon-IGS interaction is already amply supported by experimental evidence, we concentrated our efforts on testing the involvement of the 3' exon nucleotides in the selection of the 3' splice site. We introduced a number of substitutions in the IGS and 3' exon of aI3 (429)/wt as illustrated in figure 3BD. For comparison the wild type interactions are shown in figure 3A. The U19A:U20A mutation in the IGS disturbs both the interaction between the first two intron nucleotides and the IGS

and the possible interaction between the 3' exon and the IGS (fig. 3B). The second mutation (A430U:A431U) disturbs the potential interaction between the 3' exon and the IGS but leaves the interaction between the 5' splice site and the IGS unchanged (fig. 3C). A combination of both mutations restores the potential interaction between the 3' exon and the IGS but prevents basepairing of the IGS with nucleotides 1 and 2 of the intron (fig. 3D). If 3' splice site selection does not strictly depend on

3900 Nucleic Acids Research, Vol. 20, No. 15 UUGGUAACGAGAUUA MU

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AACG AGA U UA DUUG ACAUAAUU -AA AAUG U I* Figure 2. Control reactions of pT7 aI3 (429)Iwt and pT7 a13 (429)/GGGU RNA. (A) Reactions of pT7 aI3 (429)/wt. (B) Reactions of pT7 a13 (429)/GGGU. I: input; R: 20' reaction of [cs32P]-UTP labelled precursor RNA; G: 20' reaction of unlabelled precursor RNA in the presence of [ca32P]-GTP. Products are indicated with cartoons. Breakdown products of RNA molecules already present in the input lanes are indicated with an asterisk. They are not the result of selfsplicing activity. M: marker lane (Hinf I digested pBR322: 1631, 517, 506, 396, 344, 298, 221, 220 and 154 nt).

Figure 3. Mutant constructs. (A) IGS-exon alignments for pT7 a13 (429)/wt as in figure 1. (B) in the IGS mutant, U'9U20 is substituted with AA. (C) in the 3' exon mutant, A430A431 is substituted with UU. (D) the double mutant contains both changes. shaded nucleotides: exon sequences; outlined nucleotides: IGS. most likely basepairings are shown with solid lines, alternative pairings with dashed lines. Sites that were mutated are shown in rectangles. The same series of mutants was constructed for pT7 a13 (429)/GGGU.

3' exon binding to the IGS, one would expect 3' splice site selection to occur properly in all constructs. Otherwise 3' splice site selection should only occur in the wild type form and the double mutant. An analysis of splicing behaviour of RNA transcribed from the pT7 a13 (429)/wt construct and its three mutant versions is presented in figure 4A. Aliquots from three time points (lanes 2, 3 and 4 and products end-labelled by [a&32P]-GTP (lane G) are shown. (Some short transcripts indicated with an asterisk arose during the transcription reaction and are already present in the input lanes. Since the intensity of these products does not change in the course of the reactions, they do not participate in the self-splicing reaction and will not be further discussed here.) In the IGS mutant (U19A:U20A) only a very small amount of normal ligation product has been detected. Instead, another slightly shorter product is found. Sequence determination of this product revealed that it consists of the 5' exon of which the last 6 nucleotides are missing ligated to the 3' exon (results not shown). At the position of the excised intron, three GTP labelled products emerge, each with an intensity lower than that of products derived from the unsubstituted construct. The longest product could be identified as a the excised intron joined to the last 6 nucleotides of the 5' exon (results not shown). The middle

product is the normally excised intron. The nature of the smallest product could not be determined. The results of the ligation product and the excised intron point to the existence of a cryptic splice site in the 5' exon, 6 nucleotides upstream of the normal 5' splice site. This is confirmed by virtual absence of the free 5' exon and the intron-3' exon intermediates. Instead a 5' exon product lacking the last 6 nucleotides is present in large amounts. The GTP labelled intron-3' exon intermediate has 6 extra 5' exon nucleotides, as was determined by sequencing of the isolated product (results not shown). Two side products are conspicuously absent: the free 3' exon and the 5' exon-intron product of which no cryptic equivalents were found. The last aberrant product, migrating a little slower that the 3' exon, consists of the 3' exon to which the last 6 nucleotides of the 5' exon have been ligated. Its formation is not a direct consequence of GTP attack at the cryptic splice site since a normal ligation junction is present (see also discussion). The lack of free 3' exon and 5' exon-intron side products shows that direct recognition of the 3' splice site does not take place. Presence of the 3' exon in various ligation products shows that the 3' splice site can only be recognized in ligation reactions although a cryptic 5' splice site is preferred in the first step of the reaction of this mutant. Splicing products of RNA transcribed from the 3' exon mutant

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Figure 4. Self-splicing reactions of mutant RNAs. (A) Mutant series of pT7 aI3 (429)/wt. Panel A: time course of [cr32P]-UTP labelled wild type construct; Panel B: IGS mutant; Panel C: 3' exon mutant; Panel D: double mutant. 1: input RNA; 2: 5' reaction; 3: 10' reaction and 4: 20' reaction; G: 20' reaction of unlabelled RNA in the presence of [a 32P]-GTP. Products of pT7 aI3 (429)/wt are indicated with cartoons on the left and with dashed lines on the right. Additional products formed by the mutant constructs are indicated with cartoons on the right. RNA species present before initiating the self-splicing reaction are indicated with an asterisk. Unidentified products are indicated with a question mark. For clarity, the size of the free 5' exon and the 5' exon in the ligation product have been indicated. (B) Mutant series of pT7 a13 (429)/GGGU. Panels: as in A. I: input pT7 a13 (429)/GGGU transcript. R: 20' reaction with [a32P]-UTP labelled RNA; G: 20' reaction of unlabelled RNA with [c&32P]-GTP. Question mark: Unidentified products. M: marker lane (Msp I digested pBR322: 622, 527, 404, 309, 242, 238, 217, 201, 190, 180, 160 (2x) and 147 nt (2x)).

3902 Nucleic Acids Research, Vol. 20, No. 15 construct (A430U:A43 lU) are shown in figure 4A, panel C. The only products also found in the self-splicing reaction of the

unmutated construct are the 5' exon and the GTP labelled intron-3' exon intermediate. This shows that the 5' splice site is recognized normally. The low level of reaction at the 3' splice site results in a ligation product containing the entire 5' exon. Inefficient recognition of the 3' splice site also results in the absence of linear and circular intron products. Direct recognition of the 3' splice site in the hydrolysis reaction does not take place at all. Apart from these products others were observed but these were not investigated in detail (indicated with a question mark in fig. 4A; panel C). The double mutant (U19A:U20A:A430U:A431U) has aspects of both the wild type RNA and the IGS mutant (fig. 4A, panel D). The 5' splice site is opened as indicated by the formation of intron-3' exon intermediate, excised linear intron, free 5' exon and the exon ligation product. However, the cryptic 5' splice site is preferred over the normal 5' splice site. The 3' splice site is also opened as judged from the formation of free 3' exon; ligated exons and linear and circular intron.

Removal of the cryptic 5' splice site restores preference for the wild type 5' splice site To exclude the involvement of the cryptic 5' splice site in the self-splicing reactions which interferes with the use of the wild type 5' splice site, we reduced the size of the 5' exon to 4 nucleotides in a new set of mutant constructs derived from pT7 a13 (429)/wt (pT7 a13 (429)/GGGU,fig. IA and B). RNAs derived from these constructs have the same extent of 5' exonIGS basepairing as pT7 a13 (429)/wt RNAs (fig. 1B). They react in the same way as the pT7 a13 (429)/wt series apart from the use of the cryptic splice site (fig. 2B). In the reaction of pT7 a13 (429)/GGGU transcripts (fig. 4B panel A), the normal self-splicing products and intermediates are found. GTP attack at the 5' splice site is not directly detectable in lane R since the 5' exon is too small to be recovered on the gel and the length of the corresponding intron-3' exon intermediate is almost indistinguishable from that of precursor RNA. However, it can be distinguished from precursor RNA by virtue of its labelling with GTP (lane G). The ligation product is slightly larger than the free 3' exon and present in large amounts. The linear and circular intron products are also found. Unfortunately the 5' exon-intron intermediate can not be distinguished from the linear intron. The IGS mutant (fig. 4B, panel B) produces the same amount of ligation product, linear and circular intron as the pT7 a13 (429)/GGGU transcript with wild type IGS. The ligation product is the same as that found for the unsubstituted construct, so the wild type 5' splice site was used in the reaction. Free 3' exon is not formed and we therefore conclude that this mutant is not able to select the 3' splice site independently of the ligation reaction. In contrast to the pT7 a13 (429)/wt RNAs, RNA from the 3' exon mutant of pT7 a13 (429)/GGGU is almost unreactive (fig. 4B, panel C). Only very small amounts of intron-3' exon intermediate, linear and circular intron and ligation product are found. Reaction products formed by the double mutant RNA is similar to that unmutated pT7 a13 (429)/GGGU RNA. The main difference is the decreased overall reactivity (Fig. 4B, panel C). Taken together the wild type GGGU and the double mutant RNA react as expected: both the 5' and 3' splice sites are recognized faithfully in the ligation reaction albeit less efficient in the double mutant. The 3' splice site is also recognized in a

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GTP dependent cleavage reaction, but in the double mutant, hydrolysis at the 3' splice site is also observed. In the IGS mutant the 3' splice site is recognized efficiently in the ligation reaction but not in the hydrolysis reaction. Finally, the 3' exon mutant shows almost no reactivity towards the 3' splice site. Only a very small amount of ligation product was found. Production of linear and circular intron products is in agreement with these findings. These products are prominently present in the wild type; IGS mutant and double mutant constructs, but hardly detectable in the 3' splice site mutant.

DISCUSSION Two models for the selection of the 3' splice site by group I selfsplicing introns have been put forward. The original proposal by Davies et al. suggests that the 3' exon nucleotides downstream of the 3' splice site can basepair to the IGS bringing the 3' splice site in close proximity to the 5' splice site basepaired next to it. Alternative interactions to position the 3' splice site close to the reactive centre of the intron have been suggested by Burke et al. (17). This involves basepairing of two intron nucleotides preceding the 3' terminal guanosine residue with nucleotides immediately downstream of the conserved sequence element S. For a number of introns however, this interaction is weak (only one basepair) or entirely lacking, suggesting that its contribution to 3' splice site selection is not essential. For example for the a13 intron of S. cerevisiae mt DNA, there is only one A'U basepair possible. This makes this intron an interesting subject to study the two models. Unfortunately, study of group I self-splicing introns from mitochondrial genes from Saccharomyces cerevisiae is usually hampered by their size and side reactions. Due to the fortunate arrangement of essential sequence elements in the a13 intron we were able to construct a deletion mutant that lacks 1085 nucleotides of the wild type sequence. This pT7 a13 (429)/wt construct consists of only 429 nucleotides of the 3' part of the intron, and contains all necessary structure elements flanked by the wild type exon sequences. The interactions of the splice sites

Nucleic Acids Research, Vol. 20, No. 15 3903

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circularization.

with the IGS that are predicted in the splice guide model of Davies and the intron is indeed still able to perform the self-splicing reaction in vitro. We tested the splice guide model with mutation analysis. The mutations that we introduced were directed at preventing the interaction of the 3' splice site with the IGS, but this has also implications for a possible interaction between the IGS and the first three nucleotides of the intron as was outlined in the results section. Analysis of self-splicing products of the IGS mutant of pT7 aI3 (429)/wt revealed that the extended P1 interaction between the first 3 intron nucleotides and the IGS exists since destruction of two basepairs in this interaction almost completely abolishes 5' splice site recognition (fig. SA). Instead, a cryptic splice site, 6 nucleotides upstream in the 5' exon is activated. The use of this splice site restores the interaction of intron nucleotides with the IGS because it compensates the mutation in the IGS (fig. SB). The small amount of correctly ligated exons in the reaction of the IGS mutant indicates however that in the absence of the 5'-intron IGS interaction, recognition of the 5' splice site is still possible. This is also supported by the presence of a ligation product which contains 6 nucleotides of the 3' part of the 5' exon joined to the 3' exon. Most likely this product is formed when the cryptic ligation reaction stops after the first step (GTP attack at the cryptic 5' splice site) and the intron-3' exon intermediate carrying the extra six 5' exon nucleotides then proceeds in a normal ligation reaction i.e. GTP attack at the wild type 5' splice site followed by ligation to the 3' exon. To avoid the use of the cryptic 5' splice site we constructed a new series of mutants in which the size of the 5' exon was reduced to 4 nucleotides. The results obtained with this construct are not complicated by the appearance of side products. et al. are unchanged

Weakening of the PlO interaction by introducing a mutation in the IGS did not impair the selection of the 3' splice site in the ligation reactions irrespective of the length of the 5' exon. In the pT7 aI3 (429)/wt construct ligation is not as efficient as in the unmutated version but this must be attributed to the use of the cryptic 5' splice site since ligation efficiency is restored when the cryptic 5' splice site is deleted. The fact that ligation proceeds more or less normally with the IGS mutants, contradicts the splice guide model and might support the model of Burke since the P9.0 interaction is left intact. On the other hand, weakening of Pl0 by introducing a mutation in the 3' exon does impair 3' splice site selection. This is best demonstrated by the 3' exon mutation in the pT7 aI3 (429)/GGGU construct which is virtually unreactive. These apparently contradicting results can be reconciled by assuming a competition between the P1 extension and the 3' exon for binding with the IGS. In the IGS mutants both the extension of P1 and the PIO interactions are affected (fig. 3B). We expect that in this case a short PIO pairing consisting of one G3C and one A3U basepair is stronger than the remaining possible G3C pair in the extension of P1. The PIO interaction will therefore still occur after opening of the 5' splice site not being inhibited by the G3C pair in the P1 extension. Although the interaction is weak and the first two nucleotides of the 3' exon are not basepaired to the IGS we propose that it is responsible for the ligation reaction. There are at least two other cases in which the P1O interaction does not immediately start after the 3' splice site. In the Tetrahymena intron the first nucleotide of the 3' exon is not involved in a basepairing interaction with the IGS (4, 7). Although the alternative interaction involving P9.0 can compensate this defect, exon ligation is still possible even when the P9.0 interaction is destroyed (18, 19). The second example was found in a mutant version of the 21S rRNA intron from Saccharomyces cerevisiae (22). In this case there is a spacing of -four nucleotides between the 3' splice site and the PIO basepairing interaction. This makes it conceivable that the two basepair interaction in PlO of the IGS mutants is responsible for selection of the 3' splice sites in the ligation reaction. The same is true for the double mutant in which the PIO pairing is even stronger than in the IGS mutant (fig. 3D). In the 3' exon mutant however there is still a 3 basepair P1 interaction which can prevent binding of the 3' exon, even after opening of the 5' splice site (fig. 3C). This could explain why in the 3' exon mutants almost no ligation product is found. The mechanism by which the 3' splice site is hydrolysed or directly attacked by GTP can also be explained by basepairing interactions with the IGS (23). For these reactions to occur it is assumed that not the 5' splice site but the 3' splice site is basepaired to the IGS in the precursor molecule. Nucleophilic attack at the 3' splice site produces a 5' exon-intron intermediate and a free 3' exon. Direct attack of the 3' splice site by either GTP or OH- in the wild type and double mutant constructs is likely since the PlO interactions of these constructs consist of 4 basepairs that can compete with the alternative P1 extension. In the case of the IGS mutant this side reaction is unlikely since this would require a PlO interaction of two basepairs to be stronger than a four basepair extended P1 interaction although the latter one has a two nucleotide bulge. The behaviour of the 3' exon mutant can be explained if we follow the same line of reasoning. In this case the extended P1 interaction is not destroyed at all so the weakened PlO interaction will not be able to displace it. This will lead to the inability to recognize the 3' splice site

3904 Nucleic Acids Research, Vol. 20, No. 15 in both the ligation reaction and in direct attack of the 3' splice site as was found in our experiments. At this stage it can not be excluded that non Watson-Crick basepairing interactions between nucleotides around the 3' splice site and the intron play a role in the recognition of the 3' splice site. It is however unlikely that the P9.0 interaction plays a role in the selection of the 3' splice site since a functional P9.0 interaction would have resulted in 3' splice site recognition in the 3' exon mutant. Our results can be summarized in a more detailed description of the group I self-splicing reaction following the principles of the splice guide model. In the initial stage of the reaction there is a strong interaction between the 5' splice site and the IGS and no interaction with the 3' splice site (fig. 6A). After GTP attack at the 5' splice site the 6 nt P1 basepairing is split into two short helices of which the interaction of the 5' exon part with the IGS is the stronger one. We propose that the interaction of the first three intron nucleotides with the IGS is broken because of two effects: addition of the GTP moiety at the 5' end of the intron sterically hinders this interaction and the alternative interaction between the 3' exon and the IGS is stronger because it consists of more basepairs. Taken together, the 3' splice site displaces the 5' end of the intron from the IGS and the second step of the self-splicing reaction can take place (fig. 6B). After this ligation the ligated exon product is released from the intron (fig. 6C). It has been proposed by Pleij et al. that the simultaneous binding of the 5' and 3' exons to the IGS leads to a pseudoknot structure (24). In this structure, the 5' exon-IGS and 3' exonIGS interactions are composed of two short helical segments that are stacked on top of each other which yields a quasi continuous helix. The interesting feature of this structure is that the splice sites are brought in close proximity to each other in a conformation that allows splice site cleavage and exon ligation to occur with minimal conformational changes of the intron. In intron aI3 however this pseudoknot can not take place since simultaneous interaction of the 5' and 3' exons with the IGS is impossible. The intron must therefore undergo large confonnational changes as it proceeds from the cleavage reaction at the 5' splice site to exon ligation.

ACKNOWLEDGEMENTS We would like to thank Dr R.Benne for careftl reading and Mrs J.E.Vlugt-van Daalen for careful handling of the manuscript. This work was financially supported by the Netherlands Organization for Scientific Research (NWO).

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Splice site selection by intron aI3 of the COX1 gene from Saccharomyces cerevisiae.

Interactions of the 5' and 3' splice sites with intron internal sequences of the yeast mitochondrial group I intron aI3 were studied using mutation an...
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