Vol. 12, No. 2
MOLECULAR AND CELLULAR BIOLOGY, Feb. 1992, p. 696-705
0270-7306/92/020696-10$02.00/0
Connections between RNA Splicing and DNA Intron Mobility in Yeast Mitochondria: RNA Maturase and DNA Endonuclease Switching Experiments V. GOGUEL,t A. DELAHODDE, AND C. JACQ* Laboratoire de Gene'tique Moleculaire, CNRS URA 1302, Ecole Normale Superieure, 46 75230 Paris Cedex 5, France
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Received 23 August 1991/Accepted 18 November 1991
The intron-encoded proteins bI4 RNA maturase and a14 DNA endonuclease can be faithfully expressed in yeast cytoplasm from engineered forms of their mitochondrial coding sequences. In this work we studied the relationships between these two activities associated with two homologous intron-encoded proteins: the bI4 RNA maturase encoded in the fourth intron of the cytochrome b gene and the a14 DNA endonuclease (I-SceII) encoded in the fourth intron of the gene coding for the subunit I of cytochrome oxidase. Taking advantage of both the high recombinogenic properties of yeast and the similarities between the two genes, we constructed in vivo a family of hybrid genes carrying parts of both RNA maturase and DNA endonuclease coding sequences. The presence of a sequence coding for a mitochondrial targeting peptide upstream from these hybrid genes allowed us to study the properties of their translation products within the mitochondria in vivo. We thus could analyze the ability of the recombinant proteins to complement RNA maturase deficiencies in different strains. Many combinations of the two parental intronic sequences were found in the recombinants. Their structural and functional analysis revealed the following features. (i) The N-terminal half of the bI4 RNA maturase could be replaced in total by its equivalent from the aI4 DNA endonuclease without affecting the RNA maturase activity. In contrast, replacing the C-terminal half of the bI4 RNA maturase with its equivalent from the aI4 DNA endonuclease led to a very weak RNA maturase activity, indicating that this region is more differentiated and linked to the maturase activity. (ii) None of the hybrid proteins carrying an RNA maturase activity kept the DNA endonuclease activity, suggesting that the latter requires the integrity of the a14 protein. These observations are interesting because the aI4 DNA endonuclease is known to promote the propagation, at the DNA level, of the a14 intron, whereas the bI4 RNA maturase, which is required for the splicing of its coding intron, also controls the splicing process of the aI4 intron. We propose a scenario for the evolution of these intronic proteins that relies on a switch from DNA endonuclease to RNA maturase activity.
The situation concerning the group I ORFs from Saccharomyces cerevisiae (strain 777-3A) is being clarified. Three introns (bI2, b13, and bI4 in the cytochrome b gene) are not mobile and code for an RNA maturase (references 3 and 26 and the references therein). Four introns (omega in the 21S rRNA gene [16, 32] and aI4 [13, 42], a15 alpha [38a], and possibly aI3 [30a, 36] in the COX] gene) code for a DNA endonuclease, and as expected, at least three of them are mobile (omega, aI4, and aI5 alpha). We showed that p27bI4, the C-terminal 254 amino acids from the b14 intron ORF, contains the RNA maturase activity required for the RNA splicing of both b14 and a14 introns (4). Similarly, p28aI4, the C-terminal 258 amino acids from the aI4 intron ORF, has a specific DNA endonuclease activity (also termed I-SceII) which can be detected when an engineered form of the gene is expressed in Escherichia coli (13). When operating inside mitochondria, this DNA endonuclease activity promotes the propagation at the DNA level of the aI4 intron into an intronless strain (42). The aI4 intron is thus a special case of a mobile intron in which RNA splicing depends on a maturase encoded by another intron: the bI4-encoded RNA maturase. A strain deleted for the a14 intron (24) as well as the absence of mutations within the aI4 ORF that can affect mitochondrial splicing suggest that the a14 translation product is dispensable for splicing in wild-type cells. However, in vivo studies of b14 maturase deficiencies have shown that the aI4 product could be involved in RNA splicing. b14
Group I introns have several interesting features which make them attractive biological entities to understand the general questions of the role and evolution of introns. Group I introns are ubiquitous, their splicing process is always dependent on RNA architecture, and the process sometimes requires the aid of cellular factors (see references 5, 9, 16, 25, 38, and 43 for reviews). Many yeast mitochondrial group I introns contain an open reading frame (ORF) which is, in most of the cases, in frame with the preceding exon. These putative translational intron-encoded products constitute, in their C-terminal part, a family of proteins sharing similar amino acid composition and two conserved dodecapeptides (called P1 and P2 or LAGLI and DADG [18, 28, 41]). The biosynthesis and the exact role of most of these proteins are still speculative, but at least two activities are now well documented. Clearly, some of these intronic proteins are required for the RNA splicing of the intron in which they are encoded (RNA maturase activity [see reference 22 for a review]), whereas some of them are involved in intron transposition (DNA endonuclease activity [see references 16, 32, and 43 for reviews]). Interestingly, it was recently found that related proteins, encoded outside of the mitochondrial introns, could be associated with DNA endonuclease activities (29). Corresponding author. t Present address: Howard Hughes Medical Institute, Department of Biology, Brandeis University, Waltham, MA 02254. *
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maturase deficiencies can be compensated by two different suppressors. The mitochondrial suppressor mim2 is created by a single amino acid change in the aI4 ORF (15). Also, the presence of the aI4 intron ORF is required for the nuclear suppressor NAM2-1 to compensate for b14 RNA maturase
deficiencies (19). Although the bI4 RNA maturase does not display any DNA endonuclease activity under the conditions tested (13), it may nevertheless act on DNA since it has been shown to stimulate homologous recombination both in yeast mitochondria (22) and in E. coli (17). This work was devoted to the study of the structural and functional relationships between the bI4 RNA maturase and the aI4 DNA endonuclease; these two proteins share 60%o identity but carry two different activities and are involved in two different processes. For this purpose we generated, in vivo, a series of hybrid proteins. Their RNA maturase and DNA endonuclease activities were then analyzed, respectively, in yeast cells and in E. coli.
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119 MATERIALS AND METHODS Strains, plasmids, and media. All yeast strains used are isonuclear with S. cerevisiae W303-1B (MATa leu2-3 ura3-1 trpl-J ade2-1 his3-11 canl-100). The relevant mitochondrial genotype and the phenotype are represented in Fig. 1. E. coli JM101 and TG1 were used as hosts for plasmid maintenance and recovery. Minimum and complete media for growth of S. cerevisiae and E. coli were as previously described (3). Sequences of the two proteins p27 b14 and p28 a14 are shown in Fig. 2. Plasmid constructions. Plasmid YEpJB1-23-2 was used to express maturase b14 in the yeast cytoplasm (4); the derivative plasmid pVG23-2(Nt) was obtained by introducing a NotI linker, at the ClaI site, into the middle of the maturasecoding sequence. When indicated, pVG23-2(Nt) was treated with exonuclease Bal 31, after linearization at the NotI site,
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GOGUEL ET AL.
mitochondria (13); the yeast plasmid pAD23-aI4 is similar to YEpJB1-23-aI4, and it allows the expression of the endonuclease a14 in E. coli under the control of the lambda PR promoter. This was obtained by the introduction of a doublestranded DNA fragment upstream from the mitochondrial targeting sequence. Both strands were synthesized as 64-mer oligonucleotides with the following sequences: 5'GATC CAATATCTAACACCGTGCGTGTTGACTATTTTACCT CTGGCGGTGATAATGGTTGCATGA3' and 5'GATCTCA T GCAACCAT TAT CACCGCCAGAGGTAAAATAGT CA ACACGCACGGTGTTAGATATTG3'. They contain BamHI and BglII restriction sites which permit the introduction of the fragment into plasmid YEpJB1-23-aI4. pAD23-aNt was derived from pAD23-a14 by first introducing a ClaI site into the equivalent position of the maturase sequence by directed mutagenesis and then introducing a NotI linker at the ClaI site. pGPA and pUC19-A4-A5 (13) were used, respectively, to express proteins and to test for their endonuclease activity in E. coli. The plasmid pGPA4-A5 is a derivative of pGPA in which the PvuII fragment of pUC19-A4-A5 was cloned into the unique BglII site after filling in the ends. Yeast- transformation and construction of hybrid genes. Transformation of S. cerevisiae was performed by the lithium acetate method of Ito et al. (21). To 100 ,ul of competent yeast cells was added a mixture containing 1 ,ug of plasmid linearized at the NotI site, 0.5 to 2.5 ,ug of a restriction fragment, and 40 ,ug of sonicated salmon sperm DNA. Transformants were selected as LEU+ cells. Ninety-five percent of the transformants were due to a recombinational repair event between the plasmid and the vector. Growth rates. Four tester yeast strains that were defective in maturase activity were transformed with the different recombinant plasmids. Cells were grown at 28°C on N3 medium (3) containing 2% glycerol. Growth rates were followed by measuring the A600 Oligonucleotide screening. Screening of recombinant plasmids was done by colony hybridization. Plates with bacterial colonies were covered with nitrocellulose filters which were then put on new lawns to allow overnight growth. Filters were then treated as described in reference 13; nucleic acids were linked to the nitrocellulose by UV light in a Stratagene Stratalinker 1800. Filters were hybridized with 5'-end-labelled oligonucleotides. DNA sequencing. Plasmids resulting from recombination in yeast cells were extracted from the transformed yeast cells and amplified in E. coli. Single-strand DNAs were prepared by taking advantage of the fl origin of pVG23-2Nt and pAD23-aNt, and sequences were determined by the dideoxy chain termination method (35). RNA analyses. Mitochondrial RNAs from cells grown on galactose were obtained and fractionated on agarose-formaldehyde gels as described in reference 23. The RNAs were transferred to nitrocellulose filters and hybridized to nicktranslated probes prepared either from plasmid pBHWR200, which contains an intronless version of the cytochrome b gene, or from plasmid pHB17, which contains parts of exon 4 and intron 4 of the coxl gene (BamHI-HindIII fragment). RESULTS In vivo construction of the bI4/a14 hybrid genes. The experiments to be described involve the construction of a new family of genes generated by in vivo recombination of the two genes coding for the b14 RNA maturase and the a14 DNA endonuclease. This was accomplished by taking ad-
MOL. CELL. BIOL.
vantage of both the similarities between the two genes and the highly recombinogenic properties of yeast (30, 40). We used the previously engineered versions of the two genes in which the codons UGA, AUA, and CUN were changed to their universal code equivalents UGG, AUG, and ACN (3, 13). Only the first two AUA codons of the a14 coding sequence have not been changed to AUG. The corresponding protein, p28aI4, has, nevertheless, a DNA endonuclease activity which is similar to that of the mitochondrially synthesized protein (13). The presence of a sequence coding for the 12 N-terminal amino acids of the 70-kDa outer mitochondrial membrane protein (20) fused to the two intronic coding sequences allowed the translational products to be imported into the mitochondria as previously described
(4).
The experimental scheme is illustrated in Fig. 3. Two complementary approaches were used in which the plasmidencoded gene was either the RNA maturase-coding sequence (Fig. 3A) or the DNA endonuclease-coding sequence (Fig. 3B). Ends generated by double-strand breaks in a yeast plasmid are highly reactive for homologous recombination (30, 40). We used this property to introduce, by a recombinational gap repair approach, aI4 sequence fragments into the homologous b14 sequence or vice versa. This approach of manipulating in vivo plasmid-borne genes has already been shown to be very efficient (27, 33). Classification of the hybrid genes. More than 350 plasmids from yeast transformants (of strain CW02; see below), which were mostly respiration competent, were subcloned in E. coli and analyzed by colony hybridization with a set of discriminating oligonucleotides. This revealed the presence of five structural classes of hybrid genes of which 50 were completely sequenced and 29 turned out to be different (Fig. 4). Basically, genes that form classes A and B were generated by recombination between the plasmid-encoded b14 gene and the a14 gene fragment (Fig. 3A), whereas genes from classes D and E were generated by the reciprocal procedure (Fig. 3B). Class C proteins were obtained by both approaches. From the overall results it appeared that 30 fragments common to the two genes (Fig. 5) could be used as crossover points to generate hybrid genes. These crossover points are scattered all along the genes, and in addition, many of the genes sequenced appeared to be identical, which suggested that we obtained an exhaustive view of the possibilities to form hybrids between the two genes by this type of experimental approach. Phenotypes conferred by the hybrid proteins. We previously showed that the ability of the imported protein to restore respiration was correlated to its ability to restore splicing (3, 4). Therefore, RNA maturase activities of the hybrid proteins were estimated by their ability to restore respiration in several strains harboring endogenous RNA maturase deficiencies.
Once characterized, the different plasmids were used to transform four tester yeast strains (see Materials and Methods). None of these strains are able to grow on glycerol because they lack b14 RNA maturase activity and consequently are deficient in mitochondrial splicing. Strains CW01 and CW06 contain, respectively, mutations V328 and G1659, which both lead to the creation of a nonsense codon in the b14 RNA maturase-coding sequence (14). Mutation G1659 was one of the first discovered mutations that demonstrated the pleiotropic activity of the b14 RNA maturase. Growth of these two strains on glycerol could be observed only if the imported hybrid proteins were able to restore splicing of
YEAST MITOCHONDRION RNA MATURASE AND DNA ENDONUCLEASE
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glycerol). The different plasmids were then purified in E. coli, classified by oligonucleotide screening, and DNA sequenced before properties of the different proteins were assessed in the different yeast tester strains.
both introns a14 and bI4. Strain CW02 contains an intronless form of the cytochrome b gene (24) in which the aI4 intron is not spliced because of the total absence of the endogenous bI4 maturase. Strain CW05 (14a) contains mutation G1659 and is cleanly deleted for the a14 intron; this strain does not grow on glycerol because of a block in splicing of the b14 intron. Thus, CW02 and CW05 allowed us to analyze separately the splicing of either the aI4 or the bI4 intron. We examined the in vivo properties of 29 hybrid proteins representing the five classes that differ in their ability to complement bI4 RNA-maturase deficiencies (Fig. 6). Growth rates on glycerol of the different strains expressing the hybrid proteins were compared with those of the same strains in which the wild-type form of the b14 RNA maturase was cytoplasmically translated and imported into the mitochondria (doubling times on glycerol of 3 h). When the experiment was conducted with the different hybrids, the doubling time on glycerol of the transformed strains varied from 3 h to more than 12 h. In general, growth phenotypes of the different transformants were similar for proteins belonging to the same class. Class A proteins correspond to the insert of small portions of the a14 endonuclease in the middle of the b14 maturase (Hi to H8, Fig. 4). They behaved similarly if not identically to the wild-type protein in strain CW02 as well as in the three other tester strains (Fig. 6). This means that the central part of the b14 maturase (amino acids 67 to 131) could be functionally replaced by the equivalent region from the aI4 endonuclease without affecting the maturase activity. These data are consistent with the fact that this part of the protein is highly conserved: only 9 of 81 amino acids are different between the two proteins (Fig. 4). That hybrid proteins could
complement maturase mutations strongly suggested that they had RNA maturase-like properties. To obtain direct evidence of their activities, we examined mitochondrial RNAs in strain CW06, in which a cytoplasmically translated hybrid protein was targeted to the mitochondria (Fig. 7). Growth on glycerol of strain CW06 harboring either H8 or p27bI4 was identical, and as expected, H8 and the wild-type maturase gave similar relative levels of mRNAs and premRNAs. Class B proteins (H1O to H15, Fig. 4) have b14 maturase sequences in their N-terminal half and aI4 endonuclease sequences in their C-terminal half. They had a very weak ability to complement bI4 RNA maturase deficiencies. When transformed with plasmids expressing proteins H10 to H14, strain CW02 grew slowly on glycerol, with a doubling time of about 12 h at 28°C, whereas no growth was detected after 4 days for the strain harboring H15 (one additional amino acid change, Gly-114 to Asn, compared with H14; Fig. 4). This poor ability to grow on glycerol could be correlated with the low efficiency of RNA splicing. For example, H10 conferred on strain CW06 a slower growth rate on glycerol, and correlatively, the level of mRNAs was considerably reduced (Fig. 7). This was especially clear for cytochrome b mRNAs. In the case of subunit I of the cytochrome oxidase, we could detect unexpected RNAs species migrating between the precursors and the mRNAs, suggesting that H10 could induce aberrant splicing processes. Class C proteins (H16 to H22, Fig. 4) were symmetrical to class B proteins; they had an N-terminal half from the DNA endonuclease and a C-terminal half from the RNA maturase. This had an important effect on the RNA maturase activity of the proteins. While all class B proteins had a very weak
MOL. CELL. BIOL.
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RNA maturase activity, most of class C proteins behaved like the wild-type RNA maturase. Five of six proteins could restore respiration as well as what was observed with the wild-type imported maturase in strain CW02 (Fig. 6). These data indicate that the C-terminal halves of the a14 endonuclease and the bI4 maturase are functionally divergent. This observation was reinforced by a comparison of the phenotypes obtained by the importation of H15 (class B) and H16 (class C). They are perfectly symmetrical hybrid proteins. While H15 (bI4/aI4) was not able to complement any of the maturase deficiencies tested, H16 (aI4/bI4) behaved like the wild-type form of the maturase. In this structural group, H19, which has no maturase activity, is only 3 amino acids different from the active hybrid H16 (Fig. 4). Taken at face value, this would mean that these 3 amino acids play a critical role in the maturase activity. This seems to be in contradiction to the above observation that in class A hybrids the central region appears to be functionally equivalent between the two proteins. Explanations may reside either in the fact that the two types of comparisons are made
in different protein contexts or in some trivial properties of the H19 hybrid, such as protein instability. Class D proteins (H23 to H27, Fig. 4) resulted from the approach which aimed at inserting RNA maturase-coding sequence fragments into the DNA endonuclease-coding sequence. This led to proteins that had DNA endonuclease fragments at their N- and C-terminal ends. Unlike the class C proteins, which differ only in their C-terminal ends, none of the class D proteins were able to restore respiration as well as the wild-type bI4 maturase. The most drastic example came from a comparison of H16 (class C) and H27 (class D). They are identical except for 17 amino acids in their C-terminal parts; nonetheless, H16 behaved like the wildtype bI4 maturase, whereas H27 could not restore respiration. However, as previously mentioned in the case of H16/H19, this conclusion appears to be wrong when examined in a different protein context. Thus, the hybrid H26 has lost the C-terminal part and nevertheless exhibits an RNA maturase activity. These observations may in fact reflect
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tight interactions between the different parts of each proteins. Class E genes had only small fragments of b14 maturase sequences in the a14 gene. None of the resulting proteins (H28 to H31, Fig. 4) were able to restore respiration in the three strains tested (Fig. 6). Altogether these observations indicate that the integrity of the C-terminal part of the RNA maturase is required for the wild-type RNA maturase activity whereas the N-terminal half can be replaced by the DNA endonuclease equivalent. The boundary between these two domains is difficult to assess mainly because of the highly conserved central region; but as a first approximation, one can consider H15/H16 (Fig. 4) as reflecting the general situation. Thus, amino acids 1 to 127 (P1 domain) of the b14 maturase can be replaced by the homologous part of the aI4 endonuclease, whereas the fragment from amino acids 128 to 254 (P2 domain) seems to
be more differentiated and linked to the RNA maturase
activity. Some hybrid proteins require a14 product to control bI4 splicing. Growth rates on glycerol were estimated for the different transformants at three temperatures. It appeared that except in the cases of H10 to H13 (class B), all of the hybrid proteins tested that could restore respiration at one temperature or another in one strain could restore respiration in the other strains (data not shown). H10 to H13 (class B) could restore respiration at least at 36°C in strains CW01, CW02, and CW06 but not in CW05 (Fig. 6B). In addition, hybrids H20 and H22 to H25 (classes C and D) conferred a much slower growth rate in CW05 compared with that in CW06 (Fig. 6). These two strains differ only in the presence (CW06) or the absence (CW05) of an aI4 intron, showing that the absence of the aI4 intron could abolish or reduce the capacity of some
702
MOL. CELL. BIOL.
GOGUEL ET AL.
0
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FIG. 6. In vivo properties of the hybrid proteins produced in different strains deficient in their b14 maturase activities. The four tester strains are indicated with relevant genotype (upper lines). The name of the mutation in the b14 RNA maturase-coding sequence is mentioned (V328 or G1659 or the clean deletion of either intron [A]). The ability of the corresponding imported protein to restore respiration was measured by the growth of the different transformants on glycerol. +, growth similar to that of the wild-type strain (doubling time on glycerol, 3 to 5 h); E, very slow growth (doubling time, around 12 h); , intermediate growth; -, absence of growth on glycerol plates after 4 days of incubation. Panel B shows a more detailed analysis of the phenotypes conferred by the class B proteins at three temperatures. It reveals the role of the aI4 intron in sustaining the imported RNA maturase activity (compare results between CW05 and CW06).
hybrid proteins to complement mutation G1659. This suggests that, at least in this context, the wild-type translational product of the aI4 intron could be involved in the RNA splicing process of the bI4 intron. This property seemed to be related not to a particular composition of proteins but rather to the weaker activity of the hybrid proteins that could then be rescued by the aI4 translational product. This requirement for the aI4 gene is reminiscent of previous observations which showed that the aI4 ORF product could be involved in the RNA splicing of aI4 and b14 introns (see the introduction) (15, 19). However, this is the first case in which both the b14 maturase activity (imported hybrid protein) and the mitochondrial aI4 product are required for
splicing. DNA endonuclease activity of the hybrid proteins. We wanted to know whether some of the artificially created, RNA maturase-like proteins presented in Fig. 4 had also a DNA endonuclease activity. This was tested as previously described (13) by expressing the hybrid proteins in an E. coli strain that also contained a plasmid carrying the exonic junction A4-A5, which is the target site of the aI4 DNA endonuclease. Under these conditions, the expression of the aI4 DNA endonuclease led to the formation of a linearized plasmid in vivo.
None of the hybrid proteins tested from classes C, B, and D were found to be able to cut the target sequence (Fig. 8). This is all the more surprising since some of these proteins were carrying large parts of the aI4 endonuclease-coding sequence (e.g., the H10 protein contains the 191 C-terminal amino acids of the aI4 DNA endonuclease). In addition, we took at random four hybrid proteins (H15, H29, H31, and H27; Fig. 4) which were unable to complement maturase deficiencies and examined their DNA endonucleolytic activities in E. coli. It turned out that at least one of them had an aI4-specific DNA endonuclease activity (H29; Fig. 8), and a very weak activity could also be observed in the case of H31. The H29 endonuclease is symmetrical to the H2 protein that has a wild-type maturase activity. This is another indication that a few amino acid changes in the central part of these proteins do not seem to alter their activity. These data indicate that unlike the bI4 RNA maturase, the aI4 DNA endonuclease requires most of its structure for its activity. In addition, among the 14 hybrid proteins examined, none turned out to have both activities, which suggests that these two activities could be mutually exclusive.
YEAST MITOCHONDRION RNA MATURASE AND DNA ENDONUCLEASE
VOL. 12, 1992
04
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DISCUSSI ON
We have taken advantage of soime of the recombinogenic properties of yeast to construct al large repertoire of RNA maturases that are derived from tthe in vivo recombination between the two parental homolotgous genes coding for the bI4 RNA maturase and for the aI4t DNA endonuclease (also termed I-SceII). Cotransformatio In of a linearized plasmid containing the universal code equiivalent of one of the two proteins with a DNA fragment conitaining the universal code equivalent of the other protein ((3, 13) generated a large family of recombinants. The re sulting chimeric proteins were tested for their ability to cc)mplement different mitochondrial maturase deficiencies. 'The reshuffling pattern of B
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MOLECULAR AND CELLULAR BIOLOGY, Feb. 1992, p. 696-705
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Connections between RNA Splicing and DNA Intron Mobility in Yeast Mitochondria: RNA Maturase and DNA Endonuclease Switching Experiments V. GOGUEL,t A. DELAHODDE, AND C. JACQ* Laboratoire de Gene'tique Moleculaire, CNRS URA 1302, Ecole Normale Superieure, 46 75230 Paris Cedex 5, France
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Received 23 August 1991/Accepted 18 November 1991
The intron-encoded proteins bI4 RNA maturase and a14 DNA endonuclease can be faithfully expressed in yeast cytoplasm from engineered forms of their mitochondrial coding sequences. In this work we studied the relationships between these two activities associated with two homologous intron-encoded proteins: the bI4 RNA maturase encoded in the fourth intron of the cytochrome b gene and the a14 DNA endonuclease (I-SceII) encoded in the fourth intron of the gene coding for the subunit I of cytochrome oxidase. Taking advantage of both the high recombinogenic properties of yeast and the similarities between the two genes, we constructed in vivo a family of hybrid genes carrying parts of both RNA maturase and DNA endonuclease coding sequences. The presence of a sequence coding for a mitochondrial targeting peptide upstream from these hybrid genes allowed us to study the properties of their translation products within the mitochondria in vivo. We thus could analyze the ability of the recombinant proteins to complement RNA maturase deficiencies in different strains. Many combinations of the two parental intronic sequences were found in the recombinants. Their structural and functional analysis revealed the following features. (i) The N-terminal half of the bI4 RNA maturase could be replaced in total by its equivalent from the aI4 DNA endonuclease without affecting the RNA maturase activity. In contrast, replacing the C-terminal half of the bI4 RNA maturase with its equivalent from the aI4 DNA endonuclease led to a very weak RNA maturase activity, indicating that this region is more differentiated and linked to the maturase activity. (ii) None of the hybrid proteins carrying an RNA maturase activity kept the DNA endonuclease activity, suggesting that the latter requires the integrity of the a14 protein. These observations are interesting because the aI4 DNA endonuclease is known to promote the propagation, at the DNA level, of the a14 intron, whereas the bI4 RNA maturase, which is required for the splicing of its coding intron, also controls the splicing process of the aI4 intron. We propose a scenario for the evolution of these intronic proteins that relies on a switch from DNA endonuclease to RNA maturase activity.
The situation concerning the group I ORFs from Saccharomyces cerevisiae (strain 777-3A) is being clarified. Three introns (bI2, b13, and bI4 in the cytochrome b gene) are not mobile and code for an RNA maturase (references 3 and 26 and the references therein). Four introns (omega in the 21S rRNA gene [16, 32] and aI4 [13, 42], a15 alpha [38a], and possibly aI3 [30a, 36] in the COX] gene) code for a DNA endonuclease, and as expected, at least three of them are mobile (omega, aI4, and aI5 alpha). We showed that p27bI4, the C-terminal 254 amino acids from the b14 intron ORF, contains the RNA maturase activity required for the RNA splicing of both b14 and a14 introns (4). Similarly, p28aI4, the C-terminal 258 amino acids from the aI4 intron ORF, has a specific DNA endonuclease activity (also termed I-SceII) which can be detected when an engineered form of the gene is expressed in Escherichia coli (13). When operating inside mitochondria, this DNA endonuclease activity promotes the propagation at the DNA level of the aI4 intron into an intronless strain (42). The aI4 intron is thus a special case of a mobile intron in which RNA splicing depends on a maturase encoded by another intron: the bI4-encoded RNA maturase. A strain deleted for the a14 intron (24) as well as the absence of mutations within the aI4 ORF that can affect mitochondrial splicing suggest that the a14 translation product is dispensable for splicing in wild-type cells. However, in vivo studies of b14 maturase deficiencies have shown that the aI4 product could be involved in RNA splicing. b14
Group I introns have several interesting features which make them attractive biological entities to understand the general questions of the role and evolution of introns. Group I introns are ubiquitous, their splicing process is always dependent on RNA architecture, and the process sometimes requires the aid of cellular factors (see references 5, 9, 16, 25, 38, and 43 for reviews). Many yeast mitochondrial group I introns contain an open reading frame (ORF) which is, in most of the cases, in frame with the preceding exon. These putative translational intron-encoded products constitute, in their C-terminal part, a family of proteins sharing similar amino acid composition and two conserved dodecapeptides (called P1 and P2 or LAGLI and DADG [18, 28, 41]). The biosynthesis and the exact role of most of these proteins are still speculative, but at least two activities are now well documented. Clearly, some of these intronic proteins are required for the RNA splicing of the intron in which they are encoded (RNA maturase activity [see reference 22 for a review]), whereas some of them are involved in intron transposition (DNA endonuclease activity [see references 16, 32, and 43 for reviews]). Interestingly, it was recently found that related proteins, encoded outside of the mitochondrial introns, could be associated with DNA endonuclease activities (29). Corresponding author. t Present address: Howard Hughes Medical Institute, Department of Biology, Brandeis University, Waltham, MA 02254. *
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maturase deficiencies can be compensated by two different suppressors. The mitochondrial suppressor mim2 is created by a single amino acid change in the aI4 ORF (15). Also, the presence of the aI4 intron ORF is required for the nuclear suppressor NAM2-1 to compensate for b14 RNA maturase
deficiencies (19). Although the bI4 RNA maturase does not display any DNA endonuclease activity under the conditions tested (13), it may nevertheless act on DNA since it has been shown to stimulate homologous recombination both in yeast mitochondria (22) and in E. coli (17). This work was devoted to the study of the structural and functional relationships between the bI4 RNA maturase and the aI4 DNA endonuclease; these two proteins share 60%o identity but carry two different activities and are involved in two different processes. For this purpose we generated, in vivo, a series of hybrid proteins. Their RNA maturase and DNA endonuclease activities were then analyzed, respectively, in yeast cells and in E. coli.
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119 MATERIALS AND METHODS Strains, plasmids, and media. All yeast strains used are isonuclear with S. cerevisiae W303-1B (MATa leu2-3 ura3-1 trpl-J ade2-1 his3-11 canl-100). The relevant mitochondrial genotype and the phenotype are represented in Fig. 1. E. coli JM101 and TG1 were used as hosts for plasmid maintenance and recovery. Minimum and complete media for growth of S. cerevisiae and E. coli were as previously described (3). Sequences of the two proteins p27 b14 and p28 a14 are shown in Fig. 2. Plasmid constructions. Plasmid YEpJB1-23-2 was used to express maturase b14 in the yeast cytoplasm (4); the derivative plasmid pVG23-2(Nt) was obtained by introducing a NotI linker, at the ClaI site, into the middle of the maturasecoding sequence. When indicated, pVG23-2(Nt) was treated with exonuclease Bal 31, after linearization at the NotI site,
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GOGUEL ET AL.
mitochondria (13); the yeast plasmid pAD23-aI4 is similar to YEpJB1-23-aI4, and it allows the expression of the endonuclease a14 in E. coli under the control of the lambda PR promoter. This was obtained by the introduction of a doublestranded DNA fragment upstream from the mitochondrial targeting sequence. Both strands were synthesized as 64-mer oligonucleotides with the following sequences: 5'GATC CAATATCTAACACCGTGCGTGTTGACTATTTTACCT CTGGCGGTGATAATGGTTGCATGA3' and 5'GATCTCA T GCAACCAT TAT CACCGCCAGAGGTAAAATAGT CA ACACGCACGGTGTTAGATATTG3'. They contain BamHI and BglII restriction sites which permit the introduction of the fragment into plasmid YEpJB1-23-aI4. pAD23-aNt was derived from pAD23-a14 by first introducing a ClaI site into the equivalent position of the maturase sequence by directed mutagenesis and then introducing a NotI linker at the ClaI site. pGPA and pUC19-A4-A5 (13) were used, respectively, to express proteins and to test for their endonuclease activity in E. coli. The plasmid pGPA4-A5 is a derivative of pGPA in which the PvuII fragment of pUC19-A4-A5 was cloned into the unique BglII site after filling in the ends. Yeast- transformation and construction of hybrid genes. Transformation of S. cerevisiae was performed by the lithium acetate method of Ito et al. (21). To 100 ,ul of competent yeast cells was added a mixture containing 1 ,ug of plasmid linearized at the NotI site, 0.5 to 2.5 ,ug of a restriction fragment, and 40 ,ug of sonicated salmon sperm DNA. Transformants were selected as LEU+ cells. Ninety-five percent of the transformants were due to a recombinational repair event between the plasmid and the vector. Growth rates. Four tester yeast strains that were defective in maturase activity were transformed with the different recombinant plasmids. Cells were grown at 28°C on N3 medium (3) containing 2% glycerol. Growth rates were followed by measuring the A600 Oligonucleotide screening. Screening of recombinant plasmids was done by colony hybridization. Plates with bacterial colonies were covered with nitrocellulose filters which were then put on new lawns to allow overnight growth. Filters were then treated as described in reference 13; nucleic acids were linked to the nitrocellulose by UV light in a Stratagene Stratalinker 1800. Filters were hybridized with 5'-end-labelled oligonucleotides. DNA sequencing. Plasmids resulting from recombination in yeast cells were extracted from the transformed yeast cells and amplified in E. coli. Single-strand DNAs were prepared by taking advantage of the fl origin of pVG23-2Nt and pAD23-aNt, and sequences were determined by the dideoxy chain termination method (35). RNA analyses. Mitochondrial RNAs from cells grown on galactose were obtained and fractionated on agarose-formaldehyde gels as described in reference 23. The RNAs were transferred to nitrocellulose filters and hybridized to nicktranslated probes prepared either from plasmid pBHWR200, which contains an intronless version of the cytochrome b gene, or from plasmid pHB17, which contains parts of exon 4 and intron 4 of the coxl gene (BamHI-HindIII fragment). RESULTS In vivo construction of the bI4/a14 hybrid genes. The experiments to be described involve the construction of a new family of genes generated by in vivo recombination of the two genes coding for the b14 RNA maturase and the a14 DNA endonuclease. This was accomplished by taking ad-
MOL. CELL. BIOL.
vantage of both the similarities between the two genes and the highly recombinogenic properties of yeast (30, 40). We used the previously engineered versions of the two genes in which the codons UGA, AUA, and CUN were changed to their universal code equivalents UGG, AUG, and ACN (3, 13). Only the first two AUA codons of the a14 coding sequence have not been changed to AUG. The corresponding protein, p28aI4, has, nevertheless, a DNA endonuclease activity which is similar to that of the mitochondrially synthesized protein (13). The presence of a sequence coding for the 12 N-terminal amino acids of the 70-kDa outer mitochondrial membrane protein (20) fused to the two intronic coding sequences allowed the translational products to be imported into the mitochondria as previously described
(4).
The experimental scheme is illustrated in Fig. 3. Two complementary approaches were used in which the plasmidencoded gene was either the RNA maturase-coding sequence (Fig. 3A) or the DNA endonuclease-coding sequence (Fig. 3B). Ends generated by double-strand breaks in a yeast plasmid are highly reactive for homologous recombination (30, 40). We used this property to introduce, by a recombinational gap repair approach, aI4 sequence fragments into the homologous b14 sequence or vice versa. This approach of manipulating in vivo plasmid-borne genes has already been shown to be very efficient (27, 33). Classification of the hybrid genes. More than 350 plasmids from yeast transformants (of strain CW02; see below), which were mostly respiration competent, were subcloned in E. coli and analyzed by colony hybridization with a set of discriminating oligonucleotides. This revealed the presence of five structural classes of hybrid genes of which 50 were completely sequenced and 29 turned out to be different (Fig. 4). Basically, genes that form classes A and B were generated by recombination between the plasmid-encoded b14 gene and the a14 gene fragment (Fig. 3A), whereas genes from classes D and E were generated by the reciprocal procedure (Fig. 3B). Class C proteins were obtained by both approaches. From the overall results it appeared that 30 fragments common to the two genes (Fig. 5) could be used as crossover points to generate hybrid genes. These crossover points are scattered all along the genes, and in addition, many of the genes sequenced appeared to be identical, which suggested that we obtained an exhaustive view of the possibilities to form hybrids between the two genes by this type of experimental approach. Phenotypes conferred by the hybrid proteins. We previously showed that the ability of the imported protein to restore respiration was correlated to its ability to restore splicing (3, 4). Therefore, RNA maturase activities of the hybrid proteins were estimated by their ability to restore respiration in several strains harboring endogenous RNA maturase deficiencies.
Once characterized, the different plasmids were used to transform four tester yeast strains (see Materials and Methods). None of these strains are able to grow on glycerol because they lack b14 RNA maturase activity and consequently are deficient in mitochondrial splicing. Strains CW01 and CW06 contain, respectively, mutations V328 and G1659, which both lead to the creation of a nonsense codon in the b14 RNA maturase-coding sequence (14). Mutation G1659 was one of the first discovered mutations that demonstrated the pleiotropic activity of the b14 RNA maturase. Growth of these two strains on glycerol could be observed only if the imported hybrid proteins were able to restore splicing of
YEAST MITOCHONDRION RNA MATURASE AND DNA ENDONUCLEASE
VOL. 12, 1992 f lori
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glycerol). The different plasmids were then purified in E. coli, classified by oligonucleotide screening, and DNA sequenced before properties of the different proteins were assessed in the different yeast tester strains.
both introns a14 and bI4. Strain CW02 contains an intronless form of the cytochrome b gene (24) in which the aI4 intron is not spliced because of the total absence of the endogenous bI4 maturase. Strain CW05 (14a) contains mutation G1659 and is cleanly deleted for the a14 intron; this strain does not grow on glycerol because of a block in splicing of the b14 intron. Thus, CW02 and CW05 allowed us to analyze separately the splicing of either the aI4 or the bI4 intron. We examined the in vivo properties of 29 hybrid proteins representing the five classes that differ in their ability to complement bI4 RNA-maturase deficiencies (Fig. 6). Growth rates on glycerol of the different strains expressing the hybrid proteins were compared with those of the same strains in which the wild-type form of the b14 RNA maturase was cytoplasmically translated and imported into the mitochondria (doubling times on glycerol of 3 h). When the experiment was conducted with the different hybrids, the doubling time on glycerol of the transformed strains varied from 3 h to more than 12 h. In general, growth phenotypes of the different transformants were similar for proteins belonging to the same class. Class A proteins correspond to the insert of small portions of the a14 endonuclease in the middle of the b14 maturase (Hi to H8, Fig. 4). They behaved similarly if not identically to the wild-type protein in strain CW02 as well as in the three other tester strains (Fig. 6). This means that the central part of the b14 maturase (amino acids 67 to 131) could be functionally replaced by the equivalent region from the aI4 endonuclease without affecting the maturase activity. These data are consistent with the fact that this part of the protein is highly conserved: only 9 of 81 amino acids are different between the two proteins (Fig. 4). That hybrid proteins could
complement maturase mutations strongly suggested that they had RNA maturase-like properties. To obtain direct evidence of their activities, we examined mitochondrial RNAs in strain CW06, in which a cytoplasmically translated hybrid protein was targeted to the mitochondria (Fig. 7). Growth on glycerol of strain CW06 harboring either H8 or p27bI4 was identical, and as expected, H8 and the wild-type maturase gave similar relative levels of mRNAs and premRNAs. Class B proteins (H1O to H15, Fig. 4) have b14 maturase sequences in their N-terminal half and aI4 endonuclease sequences in their C-terminal half. They had a very weak ability to complement bI4 RNA maturase deficiencies. When transformed with plasmids expressing proteins H10 to H14, strain CW02 grew slowly on glycerol, with a doubling time of about 12 h at 28°C, whereas no growth was detected after 4 days for the strain harboring H15 (one additional amino acid change, Gly-114 to Asn, compared with H14; Fig. 4). This poor ability to grow on glycerol could be correlated with the low efficiency of RNA splicing. For example, H10 conferred on strain CW06 a slower growth rate on glycerol, and correlatively, the level of mRNAs was considerably reduced (Fig. 7). This was especially clear for cytochrome b mRNAs. In the case of subunit I of the cytochrome oxidase, we could detect unexpected RNAs species migrating between the precursors and the mRNAs, suggesting that H10 could induce aberrant splicing processes. Class C proteins (H16 to H22, Fig. 4) were symmetrical to class B proteins; they had an N-terminal half from the DNA endonuclease and a C-terminal half from the RNA maturase. This had an important effect on the RNA maturase activity of the proteins. While all class B proteins had a very weak
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FIG. 4. Structural organization of the chimeric proteins. The two proteins p28a14 (DNA endonuclease, open bar) and p27bI4 (RNA maturase, dark bar) are schematically represented in the two upper lines. Amino acids differing between the two sequences are represented and extended as vertical dotted lines. Also represented are the two conserved dodecapeptides P1 and P2. In the different hybrid proteins, RNA maturase sequences are represented by dark bars and DNA endonuclease sequences are represented by open bars. Hybrid proteins were classified according to their general composition in five groups. Proteins from classes A and B and H16, H17, and H19 were obtained from experiment described for Fig. 3A, whereas proteins from classes D and E and H20, H21, and H22 were obtained from the reciprocal procedure (Fig. 3B). Most of these hybrid proteins have been selected for their ability to restore respiration in strain CW02 (deleted for the b14 intron), and the resulting phenotypes are mentioned on the right. Some hybrid proteins that were not able to restore respiration were also
picked up randomly (class E).
RNA maturase activity, most of class C proteins behaved like the wild-type RNA maturase. Five of six proteins could restore respiration as well as what was observed with the wild-type imported maturase in strain CW02 (Fig. 6). These data indicate that the C-terminal halves of the a14 endonuclease and the bI4 maturase are functionally divergent. This observation was reinforced by a comparison of the phenotypes obtained by the importation of H15 (class B) and H16 (class C). They are perfectly symmetrical hybrid proteins. While H15 (bI4/aI4) was not able to complement any of the maturase deficiencies tested, H16 (aI4/bI4) behaved like the wild-type form of the maturase. In this structural group, H19, which has no maturase activity, is only 3 amino acids different from the active hybrid H16 (Fig. 4). Taken at face value, this would mean that these 3 amino acids play a critical role in the maturase activity. This seems to be in contradiction to the above observation that in class A hybrids the central region appears to be functionally equivalent between the two proteins. Explanations may reside either in the fact that the two types of comparisons are made
in different protein contexts or in some trivial properties of the H19 hybrid, such as protein instability. Class D proteins (H23 to H27, Fig. 4) resulted from the approach which aimed at inserting RNA maturase-coding sequence fragments into the DNA endonuclease-coding sequence. This led to proteins that had DNA endonuclease fragments at their N- and C-terminal ends. Unlike the class C proteins, which differ only in their C-terminal ends, none of the class D proteins were able to restore respiration as well as the wild-type bI4 maturase. The most drastic example came from a comparison of H16 (class C) and H27 (class D). They are identical except for 17 amino acids in their C-terminal parts; nonetheless, H16 behaved like the wildtype bI4 maturase, whereas H27 could not restore respiration. However, as previously mentioned in the case of H16/H19, this conclusion appears to be wrong when examined in a different protein context. Thus, the hybrid H26 has lost the C-terminal part and nevertheless exhibits an RNA maturase activity. These observations may in fact reflect
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YEAST MITOCHONDRION RNA MATURASE AND DNA ENDONUCLEASE
VOL . 12 1992 ,
701
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FIG. 5. Localization of the crossover points observed in the different recombinant genes. The sequences coding for the two proteins (upper lines correspond to the p28aI4 DNA endonuclease, and lower lines correspond to the p27bI4 RNA maturase) are shown with their conserved nucleotides (black dots). Boxed sequences indicate the 30 different homologous regions where crossovers have been observed in the different hybrid genes.
tight interactions between the different parts of each proteins. Class E genes had only small fragments of b14 maturase sequences in the a14 gene. None of the resulting proteins (H28 to H31, Fig. 4) were able to restore respiration in the three strains tested (Fig. 6). Altogether these observations indicate that the integrity of the C-terminal part of the RNA maturase is required for the wild-type RNA maturase activity whereas the N-terminal half can be replaced by the DNA endonuclease equivalent. The boundary between these two domains is difficult to assess mainly because of the highly conserved central region; but as a first approximation, one can consider H15/H16 (Fig. 4) as reflecting the general situation. Thus, amino acids 1 to 127 (P1 domain) of the b14 maturase can be replaced by the homologous part of the aI4 endonuclease, whereas the fragment from amino acids 128 to 254 (P2 domain) seems to
be more differentiated and linked to the RNA maturase
activity. Some hybrid proteins require a14 product to control bI4 splicing. Growth rates on glycerol were estimated for the different transformants at three temperatures. It appeared that except in the cases of H10 to H13 (class B), all of the hybrid proteins tested that could restore respiration at one temperature or another in one strain could restore respiration in the other strains (data not shown). H10 to H13 (class B) could restore respiration at least at 36°C in strains CW01, CW02, and CW06 but not in CW05 (Fig. 6B). In addition, hybrids H20 and H22 to H25 (classes C and D) conferred a much slower growth rate in CW05 compared with that in CW06 (Fig. 6). These two strains differ only in the presence (CW06) or the absence (CW05) of an aI4 intron, showing that the absence of the aI4 intron could abolish or reduce the capacity of some
702
MOL. CELL. BIOL.
GOGUEL ET AL.
0
0
FIG. 6. In vivo properties of the hybrid proteins produced in different strains deficient in their b14 maturase activities. The four tester strains are indicated with relevant genotype (upper lines). The name of the mutation in the b14 RNA maturase-coding sequence is mentioned (V328 or G1659 or the clean deletion of either intron [A]). The ability of the corresponding imported protein to restore respiration was measured by the growth of the different transformants on glycerol. +, growth similar to that of the wild-type strain (doubling time on glycerol, 3 to 5 h); E, very slow growth (doubling time, around 12 h); , intermediate growth; -, absence of growth on glycerol plates after 4 days of incubation. Panel B shows a more detailed analysis of the phenotypes conferred by the class B proteins at three temperatures. It reveals the role of the aI4 intron in sustaining the imported RNA maturase activity (compare results between CW05 and CW06).
hybrid proteins to complement mutation G1659. This suggests that, at least in this context, the wild-type translational product of the aI4 intron could be involved in the RNA splicing process of the bI4 intron. This property seemed to be related not to a particular composition of proteins but rather to the weaker activity of the hybrid proteins that could then be rescued by the aI4 translational product. This requirement for the aI4 gene is reminiscent of previous observations which showed that the aI4 ORF product could be involved in the RNA splicing of aI4 and b14 introns (see the introduction) (15, 19). However, this is the first case in which both the b14 maturase activity (imported hybrid protein) and the mitochondrial aI4 product are required for
splicing. DNA endonuclease activity of the hybrid proteins. We wanted to know whether some of the artificially created, RNA maturase-like proteins presented in Fig. 4 had also a DNA endonuclease activity. This was tested as previously described (13) by expressing the hybrid proteins in an E. coli strain that also contained a plasmid carrying the exonic junction A4-A5, which is the target site of the aI4 DNA endonuclease. Under these conditions, the expression of the aI4 DNA endonuclease led to the formation of a linearized plasmid in vivo.
None of the hybrid proteins tested from classes C, B, and D were found to be able to cut the target sequence (Fig. 8). This is all the more surprising since some of these proteins were carrying large parts of the aI4 endonuclease-coding sequence (e.g., the H10 protein contains the 191 C-terminal amino acids of the aI4 DNA endonuclease). In addition, we took at random four hybrid proteins (H15, H29, H31, and H27; Fig. 4) which were unable to complement maturase deficiencies and examined their DNA endonucleolytic activities in E. coli. It turned out that at least one of them had an aI4-specific DNA endonuclease activity (H29; Fig. 8), and a very weak activity could also be observed in the case of H31. The H29 endonuclease is symmetrical to the H2 protein that has a wild-type maturase activity. This is another indication that a few amino acid changes in the central part of these proteins do not seem to alter their activity. These data indicate that unlike the bI4 RNA maturase, the aI4 DNA endonuclease requires most of its structure for its activity. In addition, among the 14 hybrid proteins examined, none turned out to have both activities, which suggests that these two activities could be mutually exclusive.
YEAST MITOCHONDRION RNA MATURASE AND DNA ENDONUCLEASE
VOL. 12, 1992
04
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two coding sequences that lead to new RNA maturase11-the like proteins turned out to be unexpectedly simple. The high
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level of similarity between the coding sequences for the bI4 RNA maturase and the aI4 DNA endonuclease could have led to a complex patchwork of the two types of sequences. The selected hybrids thus reflect the functional screening of the recombinants, and the mosaic compositions presented in Fig. 4 are likely to represent most of the functional combinations of the two coding sequences. Moreover, the recent discovery (2) that unstable pseudorevertants of maturaseless mutants could arise in mitochondria by similar recombinational events gives credence to our study. Protein shuffling between RNA maturase and DNA endonuclease. The high similarity shared by the two amino acid sequences might suggest a functional equivalence of the
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FIG. 7. Northern blot (RNA blot) analyses of a14 and b14 splicing. Mitochondrial RNAs were extrac3ed from strain CW06 transformed with different plasmids coding for hybrid proteins. Hybridizations were carried out with exo probes specific for the cytochrome b gene (A) or for the genie coding for the subunit I of cytochrome oxidase (B) (see Material ls and Methods). Sizes of the mRNA and pre-mRNA species are re ported on the right. Controls are p27 (wild-type imported b14 maturnase) and strain CW05 (deleted for the a14 intron).
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correspondng protens Indeed, thedifferenthybrid proteins that showed some RNA maturase activity were carrying different portions of the aI4 endonuclease that altogether represented the total endonuclease protein sequence. Such an approach should, a priori, detect parts of the two proteins that are functionally conserved. This is the case of the central fragment of the b14 RNA maturase (amino acids 67 to 131) in which there is only a 9-amino-acid difference from the equivalent aI4 DNA endonuclease sequence. At least in class A hybrids (Fig. 4) this fragment could be totally or partly replaced by its aI4 DNA endonuclease counterpart without affecting the RNA maturase activity. Similarly and unexpectedly, our approach revealed that the N-terminal half (amino acids 1 to 127) of the b14 RNA maturase could be replaced by the N-terminal half of the DNA endonuclease in spite of the fact that more than 36 amino acids are different. This is well documented with the class C proteins (Fig. 4), which exhibit no important differences in their ability to complement RNA maturase mutations (Fig. 6) while they strongly differ from each other in their N-terminal regions. This high tolerance to fragment exchanges of the bI4 RNA maturase suggests that the overall structures of the two
DISCUSSI ON
We have taken advantage of soime of the recombinogenic properties of yeast to construct al large repertoire of RNA maturases that are derived from tthe in vivo recombination between the two parental homolotgous genes coding for the bI4 RNA maturase and for the aI4t DNA endonuclease (also termed I-SceII). Cotransformatio In of a linearized plasmid containing the universal code equiivalent of one of the two proteins with a DNA fragment conitaining the universal code equivalent of the other protein ((3, 13) generated a large family of recombinants. The re sulting chimeric proteins were tested for their ability to cc)mplement different mitochondrial maturase deficiencies. 'The reshuffling pattern of B
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