Site-specific DNA endonuclease and RNA maturase activities of two homologous intron-encoded proteins from yeast mitochondria

Cell, Vol. 56, 431-441,

February

10, 1989, Copyright

0 1989 by Cell Press

Site-Specific DNA Endonuclease and RNA Maturase Activities of Two Homologous Intron-Encoded Proteins from Yeast Mitochondria A. Delahodde, V. Goguel, A. M. Becam, F. Creusot, J. Perea, J. Banroques, and C. Jacq Centre de Genetique Moleculaire CNRS 91198 GiF Cedex and Laboratoire de Genetique Moleculaire CNRS UA 238 Ecole Normale Superieure 4G Rue d’Ulm 75005 Paris France

Summary Two introns of the mitochondrial genome 777-3A of S. cerevisiae, b14 in cob and al4 in coxl genes, contain ORFs that can be translated into two homologous proteins. We changed the UGA, AUA, and CUN codons of these ORFs to the universal genetic code, in order to study the functions of their translated products in E. coli and in yeast, by retargeting the nuclear encoded protein into mitochondria. The p27bl4 protein has been shown to be required for the splicing of both introns b14 and al4. The homologous p28al4 protein is highly toxic to E. coli. It can specifically cleave doublestranded DNA at a sequence representing the junction of the two fused flanking exons. We present evidence that this system is a good model for studying the role of mitochondrial intron-encoded proteins in the rearrangement of genetic information at both the RNA (RNA splicing-b14 maturase) and DNA levels (intron transposition-al4 transposase). Introduction Translation products of intronic ORFs of the yeast mitochondrial genome have been found to be involved in different processes related to RNA and DNA transactions. The RNA maturase activity of the protein encoded by the second intron of the cytochrome b gene was first revealed by genetic studies (Jacq et al., 1980; Lazowska et al., 1980). This RNA maturase activity is essential for the splicing of the intron by which the maturase is encoded. Genetic evidence for such a system has now been obtained for several introns of the yeast cytochrome b gene (Lazowska et al., 1980, 1989; Anziano et al., 1982; De La Salle et al., 1982; Weiss-Brummer et al., 1982) and for the first intron of the coxi gene encoding subunit I of cytochrome oxidase (Carignani et al., 1983). Recently, this RNA maturase activity was confirmed by the observation that engineered forms of the intronic protein, translated in the cytoplasm, could be imported into mitochondria, where they complemented splicing deficiencies (Banroques et al., 1986, 1987). intron-encoded proteins can also be involved, at the DNA level, in the mobility of mitochondrial introns. Several lines of evidence indicate that, in mitochondria or chloroplasts, introns can behave as mobile genetic elements (reviewed

by Dujon, 1981; Butow, 1985; Lemieux and Llee, 1987). The best documented example of such a mobility in Saccharomyces cerevisiae is that of the intron, which is present in the 21s rRNA gene of the omega+ strain and absent in the omega- strain (Bos et al., 1978; Faye et al., 1979; Dujon, 1980). In omega+ x omega- crosses, the high frequency conversion of the omega- to the omega+ allele requires the action of a protein encocled by the intron. This protein, called fit1 (Macreadie et al., 1985) or omega transposase (Jacquier and Dujon, 1985; Colleaux et al., 1986) has been shown to possess a specific DNA endonuclease activity cleaving the recipient omega- genome at the insert site of the omega+ intron. In addition to this spreading of organelle introns, many mitochondrial introns can be deleted genetically (Jacq et al., 1982; Labouesse and Slonimski, 1983; Gargouri et al., 1983); however, the factors controlling these deletions are not known. It has been proposed that intron-encoded proteins might play a role in this process, acting as a reverse transcriptase (Gargouri et al., 1983; Michel and Lang, 1985). In Saccharomyces cerevisiae (strain 777-3A), the fourth intron of the cytochrome b gene (b14) and the fourth intron of the gene coding for subunit I of cytochrome oxidase (a14) are both group I introns that contain an open reading frame in phase with the preceding exon (Bonitz et al., 1980a; Nobrega and Tzagoloff, 1980). In spite of their extensive structural similarities, these two intron OR’Fs have different functional properties. Genetic’studi’es have suggested, and molecular biology experiments have demonstrated, thatathe p27bl4 protein encoded in the 3’ end of the intron ORF is necessary for the splicing of both introns b14 and al4 (Kotylak and Slonimski, 1976; Dhawale et al., 1981; Labouesse et al., 1,984; Banroques et al., 1987). No direct evidence for a function of the al4-encoded product has been obtained thus far. However, following a single base change (Dujardin et al., 1982) a latent RNA maturase activity of the albencoded protein seems to be revealed. Also, an intact al4 ORF is required for the nuclear gene NAM2 to compensate for ‘bl4 maturase deficiencies (Labouesse et al., 1987; Herbert et al., 1988). On the other hand, the absence of mutations altering mitochondrial functions and localized in the al4 ORF suggiests that, in the wild-type cell, the translation product of this ORF is dispensable, whereas that of the b14 ORF is essential (Labouesse and Sloni,mski, 1983). This study focuses on the comparison of the properties of the proteins encoded by introns b14 and al4. We showed previously that engineered forms of the bl4-encoded protein can be expressed in, the ‘yeast cytoplasm and imported into mitochondria,, where they can complement maturase deficiencies (Banroques et al., 1986, 1987). In this study, we show that while the bl4-encoded protein is involved in the splicing process of b14 and ~114,the al4encoded protein can cleave dou’ble-stranded DNA at a sequence representing the junction of, the two fused exons. This suggests that RNA maturase and DNA transposase activities of intron-encoded proteins might be related.

Cell 432

B5

Bl-B4

cytb

Figure 1. Coding Regions bl4 and a14 lntrons

of the Different

Homologous

Proteins

from

Diagram showing the general features of the intron open reading frames that have been engineered to be expressed either in yeast cytoplasm or in E. coli. In the case of the fourth intron of the cytochrome b gene (upper part) from S. cerevisiae (strain 777-3A), three intron-encoded proteins have been analyzed either previously (Banroques et al., 1987) or in this work. In the case of the fourth intron of the gene coding for the subunit I of cytochrome oxidase (co.@, only one protein, p28al4 and its mutated form mim2-7, have been studied. The two homologous proteins p27bl4-2 and p28al4 possess a high degree of identity, and PI and P2 indicate the localization of two highly conserved dodecapeptides (Figure 7).

Results RNA Maturase Properties of Proteins Encoded by bl4 and al4 lntrons We recently developed a method for studying in vivo the RNA maturase activity of mitochondrial intronic proteins (Banroques et al., 1986). This method relies upon an ability to complement maturase-deficient mutations by importing into mitochondria an engineered form of the mitochondrial protein translated in yeast cytoplasm. The RNA maturase efficiency of the imported protein can be estimated by Northern blot analyses (Banroques et al., 1987) or by the growth rate of the complemented strain on respiratory substrates such as glycerol. Such an approach allowed us to demonstrate that the b14 intron-encoded proteins p27bl4-1 or a shorter form, p27bl4-2 (Figure I), can control the splicing of the introns b14 and al4 (Banroques et al., 1987). To define a minimal protein carrying the RNA maturase activity, we shortened p27bl4-2 at its N-terminal end. This produced p27bl4-5 (Figure I), which is 13 amino acids shorter. p27bl4-5 has an RNA maturase activity similar to that of p27bl4-2. Taking into account that the Pl region, which is 7 amino acids downstream of the N terminus of p27bl4-5, is essential to RNA maturase activity (Perea and Goguel, unpublished data), we assume that the minimal protein containing RNA maturase activity is 234-241 amino acids long. An unexpected finding came out of these studies. These experiments required that we convert all of the codons (UGA, AUA, and CUN) in the mitochondrial sequence to the universal genetic code (see Experimental Procedures). However, in the most active form of mRNA maturase, p27bl4-2, the CUU codon at position 202 (Figure 7) is not corrected, leading to the incorporation of a leucine when translated using the universal code. According to the proposed yeast mitochondrial genetic code

(Bonitz et al., 1980b), the CUU codon to ACU (Thr) at position 202 leads to the protein p27b14-3. It is surprising that p27bl4-3 is clearly less active than p27bl4-2. When the maturase-deficient strain CW02 was transformed with plasmids expressing p27bl4-2 or p27bl4-3, it grew on glycerol with doubling times of 4.4 hr and 6.6 hr, respectively. Two simple hypotheses can be proposed based on this observation: Either p27bl4-2 is a missense mutant of the b14 maturase, which has a higher maturase activity than the wild-type protein, or the CUU codon does not code for threonine as previously proposed, but codes for leucine as in the universal genetic code. More experiments are required to clarify this point. The p27bl-5 protein is homologous to the putative translation product of the open reading frame in the fourth intron (a14) of the coxl gene (Bonitz et al., 1980a). This raises the question of whether the homologous al4encoded protein possesses RNA maturase activity. The strategy followed to study the in vivo properties of the p28al4 protein was similar to that previously presented for the p27bl4 maturase. A universal code equivalent of the mitochondrial sequence coding for p28al4 was constructed. This necessitated introducing 23 base changes in the coding region of the protein. All of the mitochondrial codons (CUN, AUA, and UGA) were changed to their universal code equivalents by in vitro oligonucleotide-directed mutagenesis (see Experimental Procedures). The DNA sequence of the entire gene was checked after each round to ensure that no secondary mutation was introduced. The p28al4 protein, as presented in Figure 7, could thus be expressed either alone, from its own ATG, or fused to different ‘mitochondrial targeting coding sequences. Using the in vivo assay previously described, we observed that p28al4 is not able to compensate for b14 maturase deficiencies in spite of a high degree of identity between the two intronic proteins (Figure 7). An example of these results is presented in Figure 2. Such an absence of RNA maturase activity was observed under different conditions: one, when the amino acid at position 87 (Figure 7) which corresponds to a CUU codon in the mitochondrial sequence, was either a threonine or a leucine; two, when the maturase-deficient strain was deleted either for the b14 intron (strain CW02) or for the al4 intron (strain CW05; in this last case, the strain CW05 does not respire due to the presence of the mit- mutation G1659 in the b14 maturase [De La Salle et al., 1982; 6. Dujardin, personal communication]); three, when the mim2-7 mutation (glu to lys replacement at position 64, Figure 7) was introduced into the cytoplasmically translated p28al4. S’ince we know that the cytoplasmically translated .p28al4 protein is certainly imported into mitochondrima (see below and Figure 6), we have to admit that p28al4 has no RNA maturase activity. Properties of the mim2-7 mutation (Dujardin et al., 1982) have been interpreted as revealing a latent niaturase activity, possibly contained in the ~56 mrtochondrial pro-. tein, corresponding to the translation of the fused k’l to A4 exons followed by the intron 4 open reading frame. Cur observation that the p28al4-.mim2-1 protein has, no RNA maturase activity suggests that the N-terminal part of p56

Homologous 433

Intron-Encoded

Proteins

3 WO,10 G leu) Figure 2. Ability of the Different lntron Proteins in a bl4 Maturase-Deficient Strain

Glycerol to Restore

Respiration

The yeast strain CW02 is respiratory-deficient because of the absence of the bl4 maturase coding sequence in its mitochondrial genome, a result of the precise deletion of the bl4 intervening sequence (Labouesse and Slominsky, 1983). CW02 was transformed with plasmids derived from the plasmid YEpJBi-23-0 (Banroques et al., 1987) in which the different intron ORFs (Figure 1) could be fused to the mitochondrial targeting coding sequence of the 70 kd outer membrane protein (Hurt et al., 1985; Banroques et al., 1987). Transformants were first selected on a minimal medium without leucine (left), streaked on giycerol plates, and grown for 5 days at 28% (right). The different sectors represent the results of transformation with: (1) control, plasmid YEpJBi-23-0 without any intron ORF; (2) plasmid carrying the p27 bl42 coding sequence; (3) plasmid carrying the p27bl4-5 coding sequence; (4) plasmid carrying the p28al4 coding sequence; (5) plasmid carrying the p28a14-mim2-1 coding sequence.

would play a role in the pseudo-maturase activity of the mime-7 version of the al4 intron-encoded protein. This apparent absence of activity of the p28al4 protein imported into mitochondria contrasts with its unexpected activity in E. coli. p28al4 Gene Is Lethal to E. coli and Possesses a DNA Endonuclease Activity Production, in E. coli, of the two homologous proteins p27b14 and p28al4 leads to completely different effects. When placed under the transcriptional control of the lambda promoter PR, p27bl4 was expressed when the lambda repressor cl857 was denatured at 37% (Goguel et al., 1989). Under the same conditions, expression of p28al4 immediately prevented E. coli growth (lower part of Figure 3). However, when the p28al4 coding sequence was interrupted by a TGA stop codon, the corresponding truncated protein could be produced without affecting the viability of E. coli (upper part of Figure 3). An intermediate situation was found when E. coli, transformed with the expression vector carrying the p28al4 coding sequence, was first grown at 28% and then shifted to 37%. In the latter conditions, E. coli growth was immediately stopped (Figure 3). It is worth noting that E. coli strain GY7511, which permane’ntly produces ~1857, was necessary to clone the p28al4 coding sequence in an expression vector. This indicates that the presence of the cl857 coding sequence in the expression vector pGPA-al4 (Figure 4) is not sufficient to block transcription from the lambda PR

Figure 3. Production on E. coli Growth

of the p28al4

lntron Protein Has Dramatic

Effects

(0) The p28al4 coding sequence was cloned in the expression vector pGPA (Figure 4) and the E. coli strain GY7511, transformed with the recombinant plasmid at 28’%, and incubated at 37% No growth was observed. (r) The same experiment was conducted with the p28al4 coding sequence containing a TGA stop codon (position 192, Figure 7). (0 and x) correspond to growth of strains transformed with the recombinant plasmid pGPA-al4 (as in 0). The transformed strain was either grown at 28°C (0) or first grown at 28% and then shIfted to 37% (x).

promoter. Altogether, these results point out the high toxicity of p28al4 for the bacteria. Recent experiments conducted by Wenzlau et al. (1989) show that the al4 intron-encoded protein Ihas a transposase activity similar to that observed in the case of the protein encoded in the intron of the mitochondrial 21s rRNA gene of Saccharomyces cerevisiae. A double plasmid system was constructed to test, in E. coli, the effect of the synthesis of p28al4 on the DNA sequence corresponding to the fused exons A4-A5 One of tlhe two plasmids, pGPA-al4 (Figure 4), contains the coding sequence for the p28al4 protein under the control of the lambda promoter (PR). The other plasmid, pUC19-A4-A5, contains, in the polylinker region, a 30 bp sequence overlapping the junction of the fused exons. Plasmids were extracted from an E. coli-transformed strain grown either al: 28% or at 28% and shifted to 37% (where the lambda repressor is denatured), and restricted to test for the presence of a double strand cut made by the p28a14 protein. After agarose gel electrophoresis, the products weire analyzed either directly by ethidium bromide staining (Figure 4A), or by transfer to nitro&llulose and hybridization with a nicktranslated pUC19A4-A5 vector (Figure 4B). Clearly, in lane 1 of Figure 4A, we detect the presence of two DNA fragments that do not appear (lane 2) when the coding sequence for the p28al4 protein is interrupted by a UGA codon. The size of these two fragments is compatible with a doubleIstrand cut carried out by the p28al4 protein in the vicinity ‘of the junction of the two exons. This

Cell 434

A

8 Sea I

Sspl 123456

r-z3

1800

-bp bp P2s

a14

Figure

4. In Vivo Assay

of the Endonuclease

Activity

of the p28al4

lntron

Protein

The universal code equivalent of the al4 ORF was inserted in the plasmid pGPA under the transcriptional control of the lambda Ps promoter. Two coding sequences for the lambda repressor cl857 were on the same plasmid, either under the transcriptional control of its own promoter or under the control of the lac promoter. Moreover, a third copy of the gene coding for cl857 was present in the genome of the E. coli strain GM511 used in these experiments. The plasmid pUC19-A4-A5 contains the target sequence represented by the fused exons A445. This sequence is a 30 bp long synthetic oligonucleotide inserted in the EcoRl restriction site. To analyze the bans activity of p28al4 on the pUC19 A4-A5 plasmid, the appropriate E. coli strain was transformed with both plasmids that contain compatible replication origins and different markers and, after production of p28al4, the extracted DNA (see Experimental Procedures) was analyzed either directly by restriction digestion (A) or by Southern blot analyses with a nicktranslated pUCl9-A4A5 plasmid (6). (A) represents analyses by electrophoresis on agarose gel and ethidium bromide staining of an Sspl digest of the DNAs obtained when the p28al4 protein is produced either in its wild-type form (Al) or in a truncated form (A2) due to the presence of UGA codon at position 192. In (B), Southern analyses of the Seal-digested DNAs were obtained when either the p28al4 or the p27bl4-2 proteins were produced in the presence of either the A4-A5 or the 64-85 target sequences. B2:p28al4 + pUC19-A4-A5 (2W’C); 83: p28al4 + pUC19-A4-A5 (37%); 84: p28al4 + pUC19-B4-B5 (37%); 85: p27bl4-2 + pUC19-A4-A5 (37OC); 66: p27bl4-2 + pUC19-B4-B5 (37%). A3 is an EcoRI-Hindlll digest of lambda DNA. Bi is a digest of the plasmid pUC19-A4-A5 by Seal and BstEll enzymes.

was confirmed by taking advantage of the fact that the A4-A5 sequence contains a BstEll restriction site at the exon junction; the fragment generated after Sspl digestion is very similar to the Sspl-BstEll fragment obtained by double digestion of the plasmid pUC19-A4-A5 (data not shown). It is worth noting that this endonuclease activity is efficient since a large fraction of the DNA is cut even though E. coli growth is stopped shortly after transcription induction of the p28al4 gene. This point is strengthened by the observation (Figure 4, B2) that, even at 28%, when p28al4 is certainly synthesized in very low amounts, the endonuclease activity is detectable. Surprisingly, no degradation by E. coli exonucleases occurs at the cleavage site, as 5’ labeling did not reveal any heterogeneity (Figure 5). The exonic environment of the Al4 intron is very similar to that of the b14 intron (Figure 7). Thus, we can ask whether or not the p28al4 protein is able to cleave the B4-65 sequence. We inserted in the plasmid PUG19 a 200 bp fragment containing the B4-B5 junction; however, we did not detect any double strand cut when the p28al4 protein was expressed (Figure 4, 84). The possible endonuclease activity of the p27bl4 protein was examined either on the B4-B5 or on the A4-A5 junction. For this pur-

pose, the p27bl4-2 coding sequence was inserted in the plasmid pGPA under the control of’the lambda promoter. The results obtained are presented in Figure 4 (lanes B5-B6); endonuclease activity could not be detected in either case under these conditions. Sequence of the Cleavage Site Generated by the p28a14 Protein Taking advantage of the fact that the p28al4 endonuclease is highly active in E. coli and that the linearized plasmid is easily detectable in the bacteria (Figure 4) we determined the nature of the termini generated by the intronic protein directly on the DNA of an E. coli strain, transformed with both plasmids, as described in Figure 5. The purified linearized plasmid, carrying the A4-A5 sequence, was either first labeled at its 5’ termini and then cut by Pvull (Figure 5, lane b) or BamHl (Figure 5, lane c), or was first cut by Pvull (Figure 5, lane a + b) or BamHl (Figure 5, lane c+d) and then 5’ labeled. These 5’ labeled DNA fragments were compared with the Maxam-Gilbert fragments of the same DNA region. Taking into account the fact that the Maxam-Gilbert fragments migrate faster because of the presence of a phosphate at their 3’termini, and considering the sizes of the four fragments a, b, c, and

Homologous 435

lntron-Encoded

Proteins

Figure 5. Sequence of the Site Cleaved by the Maturase-like Intron-Encoded Protein p28al4

pvP II t CAGCTG GTCGAC t

BamHl ados

bp

b=los bp

1 ‘=*’ ” GGATCC d 37 b CCTAGG = P t

+

TGGTCACCCTGAA ACCAG~ACTT +

d thus obtained, we were able to determine the nature of the cleavage site cut by the p28al4 protein (Figure 5). We interpreted these results as indicating that the al4encoded protein cleaves each strand of the intronless sequence specifically, generating a 4 bp staggered cut with 3’ overhangs. To resolve ambiguities due to the presence of 3’ phosphate groups at the end of Maxam-Gilbert fragments that modify their electrophoretic mobility, we carried out the following analyses. In the first control, the plasmid pUC19/A4-A5, linearized under the action of p28al4 in E. coli, was digested with mung bean nuclease to generate flush ends from 3’hydroxyl overhangs. Religation and sequencing of the new junction confirmed the cleavage site presented in Figure 5 (data not shown). In the second control experiment, T4 DNA polymerase was added to the linearized plasmid pUC19/A4-A5 under conditions where exonuclease and polymerase activities could allow incorporation of radioactive nucleotides near the cleavage site.

Successive labeling of the cleavage site with the four dNTPs unambiguously established the nature of the cleavage site presented in Figure 5 (data not shown). The p28al4 Protein, Translated in the Yeast Cytoplasm, Can Cut the Mitochondrial Glenome We observed (see above) that the cytoplasrnically translated protein, targeted to mitochondria, has no detectable RNA maturase activity. We used the same construction to study the DNA endonuclease activity of the p28al4 protein on the mitochondrial genome. The recipient yeast strain CWO5, recently constructed (G. Dujardin, personal communication), is deleted for the intron al4 and thus contains the cleavage site recognized by p28al4. Figure 6 represents a Southern blot analysis of the mitochondrial genome of CW05 when it is transformed with a ptasmid (pYE JBI-23) carrying the coding sequence for either the wild-type p28al4 protein (Figure 6A)‘or a truncated p28al4 protein (Figure 6B). A weak but reproduciblme signal ap-

Figure 6. In Vivo Double Stranld Cut of the Mitochondrial Genome by the Cytoplasmically Translated p28al4 Protein Targeted to Mitochondria

cox I gene AB

double

stranded

DNA from the E. coli strain transformed with both plasmids pUC19-A4-A5 and pGPA-al4 (Figure 4) was extracted, and the linearized form of the plasmid pUCi9-A4-A5 was gel purified. This linear plasmid either was 5’kinase labeled (after phosphatase treatment) and digested by Pvull (b) or BamHl (c), or was first digested and then labeled at both S’ends. This last approach gave the results repesented in lane (a+b) with Pvull and inI lane (c+d) with BamHI. The different DNA fragments thus labeled were analyzed on sequencing gels and compared with the fragments generated by the Maxam and Gilbert sequencing method applied either to the Pvull labeled fragment (A) or to the BamHl labeled fragment (B) of the same plasmid. In these experiments, fragments a and d can be directly compared with Maxam and Gilbert products that have the same sequence and differ only by the presence of a 3’ phosphate group.

cut

a15,

z r

The BamHI-Xhol fragment (right) of the strain CW05 was examined for double strand breaks when the nuclear encoded p28al4 protein was targeted to mitochondria. Mitochondrial DNA from late exponential culture of the strain eO.14 kb CW05, transformed with the plasmid YEpJB l-23 (Banroques et al., 198T) carrying the p28al4 coding sequence, was purified, digested with BamHl and Xhol, fractionated on a 1% agarose gel, transferred to a nitrocellulose membrane, and hybridyzed to a nick-translated probe (dotted line) of the exon A4 (gift from P Netter). Two forms of the p28al4 protein were studied. In lane A (left), the wild-type form of the p28al4 protein was targeted to mitochondria, whereas in lane B a truncated form of the protein was produced because of the presence of a UGA codon in its coding sequence. The black arrow (right) indicates the position of the sequence recognized by the p28al4 DNA endonuclease.

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Figure 7. Sequences and p28al4

of the Two Intron-Encoded

Proteins

p27bl4-2

The upper lines represent the amino acid sequence of the p27bl4-2 protein, and the lower lines that of the p28al4 protein as they are expressed in either E. coli or S. cerevisiae cytoplasm. Black triangles indicate the position of the codons that had to be modified to adapt the mitochondrial sequences to the universal code. The corresponding oligonucleotides used for the in vitro mutagenesis are indicated in Experimental Procedures. The conserved amino acid between the two proteins are indicated with black circles.

pears with the wild-type protein that corresponds to a DNA fragment generated by a double strand cut near the homing site of the intron al4. That a specific double strand cut is generated by the p28al4 protein constitutes convincing evidence that, in this system, this protein is actually targeted to mitochondria in an active form. It is interesting that we have observed that the targeting of the p28al4 DNA endonuclease into mitochondria does not lead to a significant production of rho minus genomes, suggesting that, in this case, an efficient double strand cut repair system is active. Discussion RNA Maturase Activity of Intron-Encoded Proteins Using differently engineered forms of the open reading frames of the mitochondrial introns b14 and a14, we have studied the functions of the corresponding translation products in both S. cerevisiae mitochondria and E. coli. In yeast, the RNA maturase activity of the intron products translated in the cytoplasm and imported into mitochondria was estimated by their ability to complement mitochondrial maturase deficiencies. We have shown in this study that the protein p27bl4-5, corresponding to the 241 C-terminal amino acids of the b14 ORF, can complement

maturase deficiencies. In wild-type mitochondria, this protein is probably produced by the proteolysis of a hybrid protein encoded by exons Bl-B4 and the intron reading frame (Anziano et al., 1982; Weiss-Brummer et al., 1982). The raison d’etre of such an arrangement could be that it puts the production of the maturase under the translational control of the cytochrome b itself (Tzagoloff and Myers, 1986). In other words, both translational and posttranslational events control the box effect (Kotylak and Slonimski, 1976) by which the b14 maturase controls the splicing of both introns b14 and al4 (Dhawale et al., 1981; Labouesse et al., 1984; Banroques et al., 1987). Interestingly, the b14 maturase thus defined is homologous to the translation product of the al4 ORF. We modified all of the nonuniversal codons of the al4 sequence coding for the p28al4 protein to allow its correct translation in the yeast cytoplasm, and we fused it to a mitochondrial targeting sequence coding for the 12 N-terminal amino acids of the 70 kd outer membrane protein (Hurt et al., 1985). However, the p28al4 protein was unable to compensate for the b14 maturase deficiency under all the conditions tested. This result was unexpected because several genetic approaches had suggested that the al4 intron-encoded protein, or its precursor, could mimic the activity of the b14 maturase. This occurs when the mim2-7 mutation changes the glu residue at position 64 (Figure 7) to a lys residue (Dujardin et al., 1982). The introduction of the same amino acid change in the imported protein p28al4 did not lead to any detectable RNA maturase activity. Two extreme reasons can explain this result. Either the p28al4 protein has no activity and the maturase-like activity observed in the case of the mim2-7 mutant involves the p56 chimeric protein translated from the upstream exons and the whole intron ORF, or the p28al4 protein, imported in too large amounts into the mitochondria, alters mitochondrial functions. However, this last hypothesis is unlikely, as the import of p28al4 into wild-type mitochondria does not affect the growth of’ the transformed strain on glycerol. One could also argue that the cytoplasmically translated protein is not correctly targeted to the mitochondria, but our experiment (Figure 6) showing that p28al4 cuts a mitochondrial genome devoid of the intron al4 is good evidence that the protein can be imported into mitochondria in an active form. The Intron-Encoded Protein p28al4 Has a Specific Double Strand Endonucleolytic Activity The extreme toxicity of p28al4 in E. coli (Figure 3) was quite unexpected, since the homologous b14 maturase can be produced in E. coli, where it was shown to stimulate the homologous recombination process of the E. coli chromosome (Goguel et al., 1989). The simplest hypothesis to explain this is that the double strand endonucleolytic activity of p28al4 acts on the E. coli genome even when a very low amount of the protein is present. Such a deleterious effect is expected when a restriction endonuclease is produced in E. coli. It is perhaps more surprising in the case where few cleavages are introduced into the E. coli chromosome. Several’E. coli strains, proficient in recombination and repair, turned out to be very sensitive to p28al4

Homologous 437

Intron-Encoded

RNA (exons

Proteins

DNA

Junctions

(fused

. introns)

Junctions exons)

B4-B5

1:~$$R:fi:~:::; a

B5

c u..g.

4

production. This toxicity of p28al4 might be related to the extraordinary stability of the termini generated by its endonucleolytic activity. It can be hypothesized that such a process would be lethal if it involves the E. coli chromosome. The double strand endonucleolytic activity of p28al4 was clearly demonstrated in E. coli with a double plasmid system (Figure 4). The p28al4 protein can act in tram to cleave the DNA sequence corresponding to the A4-A5 exon junction. Few yeast endonucleases are known, but all, like p28al4, generate 4 bp extensions with 3’ overhangs (Kostriken et al., 1983; Watabe et al., 1983; Colleaux et al., 1986). There is no obvious homology between the sequences recognized by these different endonucleases, but the sequence Pl (Figure 7), which is conserved in many type I intron-encoded proteins, is also found between positions 325 and 336 in the HO endonuclease involved in the control of yeast mating type (Russell et al., 1986). The p28al4 protein is a very specific endonuclease, making a single cleavage site in the two plasmids used in this assay (Figure 4). We know from preliminary experiments that the cleavage site does not need to be larger than 24 bp (Figure 7), but a 15 bp long sequence, surrounding the cut site, is not sufficient (data not shown). Moreover, p28al4 does not cleave, in E. coli, the homologous sequence corresponding to the fusion of the two exons B4-B5 (Figure 7). However, we already know that our E. coli synthesized p28a14 protein can cleave the sequence of the recipient strain examined by Wenzlau et al. (1989), which differs at two positions in the vicinity of the cleavage site. Such a specific double strand endonucleolytic action is similar to that found for the protein coded in the rl intron of the mitochondrial21S rRNA gene of S. cerevisiae. This rl intron-encoded protein is clearly involved in a transposition process of the intron, and specificially cleaves the DNA sequence corresponding to the fused exons (Jacquier and Dujon, 1985; Macreadie et al., 1985). Concerning the al4 intron, genetic and molecular evidence

Figure 8. RNA and DNA Substrates Recognized Either by the p27bl4-2 RNA Maturase or by the p28al4 DNA Endonuclease On the left, RNA sequences of unspliced precursors are shown. Exon-intron base pairings around 5’ and 3’junctions are presented as first proposed by Waring and Davies (1984). On the right, genomic environments of b14 and al4 introns are represented. The boxed sequences represent the sequences conserved between the cytochrome b and coxl genes when the two exons 84-85 and A4-A5 are fused. The open arrows indicate the insertion site of the intervening sequences. The black arrows in the A4-A5 sequence indicate the location of the 4 bp staggered cut ‘with 3’ hydroxyl overhangs generated by p28al4. The black bar under the A4-A5 sequence points out the region that might have its structural equivalent in the RNA structure of the precursor RNA (left).

presented by Wenzlau et al. (1989) proves that a similar transposition event occurs in the case of the al4 intron. Arguments showing that this event requires the al4 translation product are presented. Their observation that a mitochondrial extract, accumulating the al4 translation product, cuts a recipient DNA, just as the engineered p28al4 protein of this study does, may indicate that the E. coli-synthesized protein retains most, if not all, of the properties of the mitochondrial al4 endonuclease. In other words, the intronic translation product corresponding to p28al4 (Figure 7) is likely to be sufficient to pn3mote, in mitochondria, the transposition process of the intron a14. Relationships between and RNA Maturases

DNA

Transposases

The proposition that maturases could have evolved from intronic transposases (Borst and Grivell, 1980) immediately followed the observation that mitochondrial introns could be considered as mobile ge;etic elements sometimes coding for RNA maturases. This idea gained important support from the finding that the rl intron protein has a specific endonucleolytic activity required for the transposition of the intron in the 21s rRNA gene (Jacquier and Dujon 1985; Macreadieet al., 1985). In that case, however, the intron-encoded protein is not required for intron splicing (Tabak et al., 1984). The results presented in this study that the two b14 and al4 intron proteins, which share extensive amino acid conservation (Figure 7), are either an RNA maturase or a DNA transposase, strongly support the view that the study of these two proteins allows an understanding of the relationship between the two properties. The prerequisite for this understanding is to elucidate the exact functions of each protein. In vitrb studies, carried out with purified forms of the p27bl4 ,maturase, have shown that it can bind to double- and single-stranded DNA as well as to RNA (Delahodde et al., 1985). The exact role of the b14 maturase in the splicing p’roces’s of both b14 and al4 introns is not known, but mutations in the b14 maturase completely block the splicing of these two gmup I introns (Banroques et al., 1987; Perea et al., unpublished data).

Cell 438

This suggests that the maturase is involved in the Ycleavage of the exon-intron junction, which is the first step of the splicing process. Considering that this 5’ cleavage is dependent upon the secondary structure of the exon-intron boundary (Waring and Davies, 1984; Perea and Jacq, 1985; Cech and Bass, 1986), we can reasonably propose that the b14 maturase is involved in the direct or indirect recognition of the double-stranded RNA structure presented in Figure 8 (left). It is interesting to point out the analogy between this RNA structure and the DNA sequence recognized by the al4 endonuclease (Figure 8), which suggests that the two proteins might act on similar signals. This similarity might explain the ability‘of the b14 maturase to have a DNA recombinase activity (Goguel et al., 1989), and the ability of the al4 transposase, in the ~56 precursor (Dujardin et al., 1982), to have a maturase activity. This constitutes an interesting situation in which a single active site recognizes both DNA and RNA helices. Finally, it is interesting to note that these two homologous intron-encoded proteins act on the two similar introns in two complementary ways. The transposase activity leads to the insertion of the intron in the genome, whereas the RNA maturase activity is involved in the RNA splicing of the intron. These observations suggest that mobile mitochondrial introns exhibit different levels of adaptation to the genomic environment. It has recently been proposed that coevolution of intron structure and intron ORFs have led to the divergence of ORF functions (Danchin, 1988). A simple form of an intron containing an open reading frame is represented by the rl intron that self-splices in vitro (van der Horst and Tabak, 1985) and encodes a protein whose function is restricted to transposition (Macreadie et al., 1985; Jacquier and Dujon, 1985). The introns bl4 and al4 represent two more elaborate steps in evolution. Neither of these introns self-splices in vitro, and they both require an intron-encoded protein for their in vivo splicing. Experimental Procedures Strains, Plasmids, and Growth Media E.coli strain

JMlOl (Alac-pro, thi, supE/ F’ tra D36, proA+B+, laclq, was obtained from New England Biolabs. Strain GY7511 (gaK,iv, his, [hclts 857, N7 N53, ABam AHI]) was given by Dr. R. Devoret. Strains used for the oligonucleotide-directed mutagenesis: TGl (K12, Ala&pro, thi, supE, hsD51 F’ traD36, proA+B+, laclq, /ad AM15); HB2151 (K12, ara14, Alac-pro, this/F’ proA+B+, laclq 1ac.Z AM15); HB2154 (same as HB2151 but mutL:TnlO); AC2522 (B/r, Hfr, sul-I) and HB2155 (same as AC2522 but mutL::TniO) were obtained from Dr. P Carter and Dr. H. Bedouelle. The strain (S. cerevisiae) CW02 used in this work is isonuclear to the strain W303-1B (R. Rothstein). The mitochondrial genome of the strain CK500/2 (intronless cytochrome b gene, constructed by M. Labouesse) has been transferred to the cell containing the nuclear genome of W303-18 by taking advantage of the kar phenotype (Conde and Fink, 1976). The resulting strain CW02 carries the appropriate markers and is efficiently transformed. The strain CW05 has been recently constructed by G. Dujardin; it is isonuclear to W303-1B and its mitochondrial genome is deleted for the intron al4 and contains the mit- mutation G1659 (De La Salle et al., 1982) in the bl4 intron. The E. coli expression vector pGPl-2 was obtained from Dr. M. Chandler and was modified as follows: The EcoRI-BamHI fragment coding for the T7 polymerase was replaced by the EcoRI-Sall fragment containing the,thermosensible repressor cl 857 and the PR promoter of phage lambda, from the pCQV2 plasmid (Queen, 1983) modified by V. Goguel. The universal code equivalent of the bl4 and al4 coding se-

/acZ AM%)

Figure

9.

quences was cloned in the unique Bgll site near the lambda promoter to produce pGPAb14 and pGPAa14, respectively. The other plasmid, pYEJBi-23, was described in Banroques et al. (1987). The plasmid pUC19A4-A5 has been constructed by inserting the syntheticoligonucleotides: 5’-AAT

TCT GAT TCT TTG GTC ACC CTG AAG TAG GA CTA AGA AAC CAG TGG GAC TTC ATC TTA A-5’

in the EcoRl restriction site of pUCI9. It should be mentioned that this synthetic sequence is identical to the A4-A5 exonic sequence only between the two restriction sites. Similarly, the plasmid PVC19 84-85 was made by inserting in pUCI9 the EcoRI-BamHI fragment overlapping the 84-85 of an intronless cytochrome b gene (see Figure 9). M13mp18 or 19 amber and M13mp18 or19 EcoK-EcoB were purchased from Anglian Biotechnology.

Synthesis and Purification of Oligonucleotides The different oligonucleotides used to adapt b14 and al4 ORFs are presented in Figure IO. Column b indicates the length of the oligonucleotides: the number of base changes (chg) are indicated. The corresponding positions of the last 3’ end base change in the oligonucleotide (codons) refer to the amino acids of the two proteins presented in Figure 7. Oligonucleotides were synthesized using an automatic DNA synthesiser (Applied Biosystems 380-A). Deprotected oligonucleotides were purified by electrophoresis in 20% polyacrylamide denaturing gels.

Oligonucleotide-Directed

Mutagenesis

The oligonucleotide-directed mutagenesis method employed was modified from Carter et al. (1985). The intron sequences were first cloned into novel Ml3 vectors (based upon M13mpl8 or 19), which carry a genetic marker that can be selected against, such as an EcoK-EcoB site or an amber mutation in an essential phage gene. In this “coupled priming” technique, one primer was used to construct the silent mutation of interest (mutagenic primer), and a second primer was used to eliminate the selectable marker on the minus strand (selection primer). After primer extension and ligation, the heteroduplex DNA was transfected into a strain of E. coli that is repair-deficient and selected against the plus strand marker (HB2151 for amber selection and HB211 or AC2522 strains for EcoK-EcoB selection). The loss of the amber marker after a round of mutagenesis was overcome with the EcoK-EcoB system,which generates a second selectable marker at the same time as removing the first one. However, in our hands, after four rounds of mutagenesis, no susceptibility to either EcoK or EcoB sites could be introduced in mutagenic phages. For the directed mutagenesis on the bl4 intron, 15 base changes were introduced with the amber selection, whereas 22 base changes were introduced with the two methods in the al4 intron sequence.

Homologous 439

Intron-Encoded

Proteins

CLlGON”CLEOTlDES (b14)

I

8],25% sucrose). The cells were resuspended in 150 ~1 of Tris-sucrose buffer containing 2.5 mglml of lysosyme and kept on ice for 10 min. After addition of 250 @I of 50 mM Tris (pH 8), 62 mM EDTA, and 0.1% Briton X-100 and incubation on ice for 10 min, the mixture was centrifuged, the supernantant made 3.75 mM with respect to ammonium acetate, and precipitated with 3 vol of ethanol. The DNA pellet was solubilized in 200 PI of water, and the insoluble material removed by centrifugation. The DNA in the clear supernatant, purified by the (GeneClean procedure (BIO 101 Inc.) was diluted in a final volume of 80 ~1. DNA Sequence Determination After each cycle of oligonucleotide-directed mutagenlzsls on both introns, the entire reading frame sequence was checked on the single stranded DNA from Ml3 recombinant using chain termination sequencing (Sanger et al., 1977). Determination of the sequence cleaved by p28al4 was made by comparing cleaved fragment products of the same DNA.

Figure

10.

Each cycle of mutagenesis included the following steps. Step 1: approximately 1 ~g of purified single-stranded DNA from the appropriate Ml3 recombinant was annealed with 25 pmols of the universal primer, 25 pmols of the mutagenic oligonucleotide, and 25 pmols of the selection primer. All of the oligonucleotides used were previously phosphorylated at their Send. Annealing was conducted in 25 VI of 30 mM *is-Cl (pH 7.6), 100 mM NaCI, and 20 mM MgC12, by slowly decreasing the temperature from 80% to room temperature. Step 2: elongation and ligation were conducted in 35 ~1 of the same buffer containing, in addition, 1 mM ATP, 10 mM of each dNTP, 10 mM dithiothreitol (pH 7.6), 2 U of Klenow polymerase (Appligene), and 5 U of T4 DNA ligase (Boehringer Mannheim). Incubation was carried out at 12°C-150C for 4 hr and then made up to 200 ~1 with 10 mM Tris, 1 mM EDTA. Step 3: The complete elongation ligation mixture was then used to transform Car&-treated E. coli cells. In the EcoK-EcoB system, considerably improved yields of mutants were obtained by taking advantage of the strains HB2154 and HB2155 (mutL::TnlO), but secondary rearrangements of sequences turned out to be more frequent. After transformation, the cells were incubated at 37% for 1 hr in 1 ml of LB medium prior to plating. Serial dilutions were then plated on LB plates using 0.1 ml of fresh E. coli cells (HB2151 or AC2522), and incubated overnight at 37°C. Screening of mutants was done by plaque hybridization. Plaques were picked up, put on new lawns, and grown overnight. These plates were covered with a nitrocellulose filter and phage diffusion was allowed for 5 min. Filters were then treated with 10% SDS for 2 min, 0.5 N NaOH11.5 M NaCl for 5 min, 0.5 M Tris-Cl/IS M NaCl for 5 min, and 6x SSC (pH 7.6) for 5 min more. Filters were baked for only 30 min at 80% in a vacuum oven and hybridized with the 5’endlabeled mutagenic oligonucleotide used for the mutagenesis. The colonies that hybridized strongly to the mutagenic oligonucleotide after a stringent wash were ,plaque-purified by toothpicking from the phage blot plate into 1 ml of LB medium. After single strand purification, the mutations were verified by dideoxy sequencing using a family of sequencing primers (mutagenic ones) located at regular intervals throughout the intron sequences. Gentle Extraction of Plasmid DNA Individual clones of cotransformants, grown overnight at 28°C in 5 ml of LB supplemented with the appropriate antibiotics were induced by a heat shock at 37% for 30 to 45 min. Control cultures were maintained at 28% for the same period of time. The cells were then collected by centrifugation and washed once in Tris-sucrose buffer (50 mM Tris [pH

Mapping of the p28al4 DNA Endonuclease Cleavage Site The E. coli strain GY7511 transformed with both plasmids, pUC19 A4A5 and pGPAa14, was grown overnight at 28°C and put at 37% for 1 hr. Supercoiled and linearized DNA were extracted, and the linearized form of pUC19 A4-A5 was gel purified. The linear plasmid was incubated at 60°C for 40 min in 50 mM Tris-Cl (pH 8.0) with 0.1 U of alcaline phosphatase (BAP, Appligene), then the enzyme was inactivated after a phenol treatment. In one case, the dephosphorylated linear plasmid was labeled at its 5’ termini with the kinase (Appligene), and digested after purification by Pvull or BamHI. In the other case, the dephosphorylated linear plasmid was first digested (Pvull or BamHI) and labeled at both 5’ends by the kinase. The different labeled DNA fragments were analyzed on sequencing gels and compared with the same fragment generated by the Maxam and Gilblsrt sequencing method. Mung Bean Digestions Th’e plasmid pEMBL 18/A4-A5, submitted in E. coli to the endonucleotyic activity of the protein p28al4, was purified as a linear plasmid and digested by mung bean nuclease in the following conditions. DNA (0.2 wg) was incubated with 2 U of mung bean nuclease (Pharmacia) in the following buffer: 50 mM sodium acetate (pH 5.0), 30 mM NaCI, 1 mM ZnS04, for 30 min at 30°C. The reaction was stopped by the addition of NaCl to 0.2 M, and DNA was purified by phenol extraction. Religated DNA was cloned in E. coli and sequenced on Its single-stranded DNA obtained by fl infection. 3’ Labeling by T4 Polymerase 3’ labeling of double strand cut generated by p28al4 was labeled by a protocol modified from Challberg and Englund (19130). Linearized plasmid (0.5 pg) was incubated with 5 U of T4 DNA polymerase (Biolabs) in 33 mM Tris acetate (pH 7.9), 66 mM potassium acetate, 0.5 mM dithiothreitol, 0.1 mglml BSA, and 10 WC of one of the four dNTPs (specific activity 400 Ci/mmols) for 25 min at 12% in a total volume of 30 ~1. The reaction was stopped by addition of 3 vol of Nal, iand the labeled DNA was purified by GeneClean. The DNA was digested with the restriction enzyme Pvull, and the size of the labeled fragments was accurately determined on a sequencing gel. Acknowledgments We are grateful to P P Slonimski for the stimulating discussions that initiated this work. We thank H. Bedouelle, P Carter, M. Chandler, R. Devoret, G. Dujardin, M. Labouesse, I? Netter, and E. Petrochillo for generous gifts of strains and plasmids. C. J. Herbert is thanked for looking over the English and for helpful discussions. We appreciated discussions with I? Perlman and R. Butow, who communicated to us prdcious unpublished data. A. D. and V. G. were supported by fellowships from the Association pour la Recherche contre le Cancer and from the MRES. This research was supported by grants from CNRS and INSERM. The costs of publication of this article were defrayed in part by the payment Of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

August

23, 1988; revised

December

16, 1988

Cell 440

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