Curr Genet (1999) 35: 109–117
© Springer-Verlag 1999
O R I G I N A L PA P E R
Georg Hausner · Claudia B. Monteiro-Vitorello Denise B. Searles · Matthew Maland Dennis W. Fulbright · Helmut Bertrand
A long open reading frame in the mitochondrial LSU rRNA group-I intron of Cryphonectria parasitica encodes a putative S5 ribosomal protein fused to a maturase Received: 7 August / 15 December 1998
Abstract A 4238-bp intervening sequence within the highly conserved U11 region of the mitochondrial large subunit ribosomal RNA gene of the fungus Cryphonectria parasitica Ep155 has been sequenced and identified to be a group-I intron. This is the largest group-I intron reported to-date for fungal mitochondrial genomes. The intron contains an 851-codon open reading frame encoding a putative, but complete, small-subunit ribosomal protein of 510 amino acids which is fused at its carboxyl terminus to a 311 amino-acid polypeptide representing a typical maturase-like protein. A short open reading frame of 83 amino acids with some similarity to maturases, but lacking a translation-initiation codon, was also noted at the 3′ end of the intron. The unusual size of the intron and the arrangement of the open and truncated reading frames suggest that this segment of the mtDNA of C. parasitica has arisen by a fusion of components from two or more different introns, possibly involving the re-location of intronic genes. Key words Cryphonectria parasitica · Mitochondrial group-I intron · Maturase · S5 ribosomal protein
G. Hausner · C. B. Monteiro-Vitorello 1 · D. B. Searles M. Maland · H. Bertrand (½) Department of Microbiology, Michigan State University, East Lansing, MI 48824-1101, USA e-mail:
[email protected] Fax: +1-517-353 8957 D. W. Fulbright Department of Botany and Plant Biology, Michigan State University, East Lansing, MI 48824-1101, USA Present address: 1 Department of Genetics, Escola Superior de Agricultura Luiz de Queiroz, University of São Paulo, 13418 Piracicaba, São Paulo, Brazil Communicated by C. W. Birky Jr.
Introduction
Mitochondrial genomes in fungi are highly variable both in size and organization. Most of this variation is due to the presence of introns and intron-encoded open reading frames (ORFs) (Wolf and Giudice 1988; Belcour et al. 1997). For example, the 62-kb mitochondrial genome of the Oak Ridge laboratory strains of Neurospora crassa contains only ten introns accounting for no more than 20 kb of DNA (Collins 1993), whereas Podospora anserina race A has a mitochondrial genome that is about 100 kb in size, of which approximately 60 kb are occupied by 36 introns and the associated ORFs (Cummings et al. 1990 b). In contrast, the mtDNA of the Ep155 laboratory strain of the chestnut-blight fungus Cryphonectria parasitica has been estimated to be at least 155 kb long, and has been predicted to consist predominantly of introns and remnants of other non-essential genetic elements (Bell et al. 1996). Mitochondrial group-I and group-II introns have been associated with maternally inherited senescence in P. anserina (reviewed by Gillham 1994), mtDNA rearrangements in yeasts caused by the mobility of some of these elements (Dujon 1989), and respiratory defects in yeast and N. crassa arising from splicing-deficiencies caused by mitochondrial- and nuclear-gene mutations (Dujon and Belcour 1989; Lambowitz and Perlman 1990; Gillham 1994). Furthermore, these introns have attracted considerable attention because some appear to have an evolutionary connection to a group of reverse-transcriptase-encoding, circular fungal mitochondrial plasmids (Lambowitz 1989; Mishra 1991). Group-I and group-II introns are classified according to their splicing pathways, their secondary structures and the presence of characteristic short conserved sequences (Lambowitz and Belfort 1993). While group-I introns seem to predominate in fungal mitochondrial DNAs, several group-II introns have also been extensively characterized (Shnyreva 1995; Yang et al. 1998). Group-II introns typically contain ORFs that code for site-specific reverse-transcriptase-like proteins. In contrast, group-I introns contain
110
ORFs which can be either free-standing within the intron or fused in-frame to an upstream exon. Group-I intronic ORFs have been shown to encode essential cellular proteins, such as the S5 ribosomal protein, site-specific endonucleases, or maturases (reviewed by Lambowitz and Belfort 1993; Shnyreva 1995). In most instances maturases promote splicing of the corresponding introns, but sometimes are site-specific endonucleases that play a role in the cleavage of DNA during the process of intron homing. Most of the maturase-like proteins contain two highly conserved dodecapeptide motifs which include conservative variants of the amino-acid sequence LAGLI-DADG (Hensgens et al. 1983; Michel and Cummings 1985; Pel and Grivell 1993). In the chestnut-blight fungus C. parasitica, an “infectious” type of hypovirulence associated with respiratory deficiencies is sometimes found in strains isolated from healing cankers on trees (Mahanti et al. 1993; D. Baidyaroy, D. H. Huber, D. W. Fulbright and H. Bertrand, unpublished observations). The role of mitochondria in the causation of this trait has been demonstrated in the virus-free, virulent Ep155 laboratory strain through the induction of mitochondrial mutations and the transmission of the associated hypovirulence phenotype from attenuated to pathogenic variants by hyphal anastomosis (Monteiro-Vitorello et al. 1995). Furthermore, the mitochondria of hypovirulent strains of Ophiostoma ulmi are usually deficient in cytochrome aa3 (Rogers et al. 1987), and contain a plasmid-like element consisting of head-to-tail concatemers of a mtDNA segment from within the LSU-rRNA gene (Abu-Amero et al. 1995). Thus, the induction of mtDNA mutations that reduce respiratory efficiency, growth rate and virulence offers a new strategy for controlling the aggressiveness of at least some of the plant-pathogenic fungi. In order to characterize mtDNA mutations that are involved in inducing hypovirulence, a physical map of the mtDNA was generated for the Ep155 strain of C. parasitica (Bell et al. 1996) as part of a search for features and factors that potentially can be exploited to generate mtDNA instabilities in this organism by in vivo and in vitro mutagenesis. In the present communication, we describe the molecular characteristics of an unusually large group-I intron that was detected in the mitochondrial LSU ribosomal DNA of this ascomycete during the mapping of its mitochondrial chromosome (Bell et al. 1996).
Materials and methods Cultures, DNA extraction and restriction endonuclease digestion. The standard Ep155 laboratory strain of C. parasitica (ATCC 38755) was the source of the mitochondrial DNA used in this study. Media, culture conditions, and the preparation of mitochondria and mtDNA have been described previously (Bell et al. 1996). DNA was digested with restriction endonucleases under conditions recommended by the manufacturer of the enzymes (Boehringer Mannheim). Restriction fragments or PCR products were separated by gel electrophoresis through 1% agarose gels in 89 mM Tris-borate buffer (with 10 mM EDTA) at pH 8.0. DNA fragments were sized using the BRL 1-kb molecular-weight ladder (Life Technologies), and the DNA
fragments were visualized by staining with ethidium bromide (0.5 µg/ml). PCR amplification, molecular cloning, DNA sequencing and analysis. The intron-containing segment of the mitochondrial LSU rRNA gene was amplified by the Expand Long Template PCR System (Boehringer Mannheim) as recommended by the manufacturer with primers IP1 and IP2, as described by Bell et al. (1996), except that 1 unit of Vent polymerase (New England Biolabs) was included in every 100 µl of the reaction mixture. The resulting 4.3-kb DNA fragment was first treated with T4 polynucleotide kinase (Life Technologies) to add terminal 5′ phosphates to the amplicons, which then were blunt-end ligated into an EcoRV-digested pBluescript™ vector (Stratagene). Standard protocols were used for the transformation of Escherichia coli DH5α and the selection of clones containing recombinant DNAs (Sambrook et al. 1989). To define the intron/exon borders within the U11 region of the mitochondrial LSU rRNA gene of C. parasitica, a library of 400 E. coli DH5α clones was generated by transforming cells with pBluescript™ (Stratagene) plasmids containing inserts of HindIII restriction fragments of C. parasitica mtDNA. The library was screened by hybridization of colony blots (Sambrook et al. 1989) with a probe generated by labeling of the PCR product of the intron with digoxigenin-dUTP using the procedure provided by the supplier of the DIG/Genius™ System (Boehringer Mannheim). Sequencing was performed on double-stranded plasmid-DNA templates purified with the Wizard™ Minipreps DNA system (Promega) using the protocol recommended by the manufacturer. The sequencing reactions were performed in accordance with instructions provided in Sequenase kits (Amersham), with the following modifications: (1) the annealing reaction, which contained about 2–3 µg of plasmid DNA and 15% formamide (Zhang et al. 1991), was boiled for 10 min and then snap-cooled in an ethanol bath at –80 °C, and (2) [α-33P]dATP (Amersham) was used for labeling the reaction products. Sequencing gels were prepared and run under conditions as outlined by Sambrook et al. (1989). The gels were vacuum-dried and exposed to Kodak X-AR film. The initial storage and analysis of the DNA sequences were performed with the programs contained within PCGENE (IntelliGenetics Inc.). Translation of the 4.3-kb insertion sequence was done in all six reading frames using the mitochondrial genetic code for ascomycetous fungi (UGA = Trp), and the three identifiable ORFs were further analyzed by computer data-base searches for sequence similarities using the Basic Local Alignment Search Tool algorithm for proteins (BLASTp, Altschul et al. 1990). The amino-acid sequences of putative, intron-encoded proteins showing >70% similarity to the hypothetical proteins encoded by the intron in the mitochondrial LSU rRNA of C. parasitica were retrieved from the GenBank data and aligned with the PCGENE CLUSTAL program. The alignments were refined visually to maximize similarity and minimize fragmentation of the aminoacid sequences.
Results and discussion
Through the use of a pair of primers, IP1 and IP2, which were designed to hybridize to the nucleotide sequences immediately upstream of and downstream form the intron that is located in the highly conserved U11 region of the mitochondrial LSU rRNA of N. crassa, we have shown previously that an insert of at least 4000 bp, presumed to be an intron, is located in the equivalent region of the single LSU rRNA gene that is located on the mitochondrial chromosome of the plasmid- and virus-free Ep155 strain of C. parasitica (Bell et al. 1996). The U11 region is one of several of the very highly conserved sequence motifs that appear in a characteristic order in LSU rRNAs (Gutell and Fox 1988). Northern-blot hybridization experiments indicated
111 Table 1 Comparison of the 5′ terminal sequence (including the putative IGS), the P, Q, R, S and 3′-end sequence of group-I introns found in the mitochondrial LSU rRNA genes of C. parasitica (C. p.), P. anserina (P. a.; Cech 1988; Cummings et al. 1989 a), N. crassa (Burke and RajBhandary 1982; Cech 1988), and P. chrysogenum (Naruse et al. 1993). For consensus sequences for the structural elements Item: C. p. P. a. N. c. P. c.
5′ terminus GTTTGTCCTTT GTTTGTCCTTC GTTTGTCCTTC TTATAACTGTGAGTCCTCC ** *****
of group-I introns see Cech (1988). Underlined regions are involved in base pairing that gives rise to the catalytic core of the group-I introns. Connecting lines below the underlined sequences indicate the potential base pairing of P with Q, of Q with R, and of R with S, to form the P4, P6, and P7 stems, respectively. Asterisks (*) denote conserved bases amongst the structural elements
P
Q
R
S
3′ end
AATTTCAAAAAT AATTTCAAAAAT AATTTCAAAAAT AATTTCAAAGAC ********* *
AATTTGAAGC AATTTGAAGC AATTTGAAGC TATTTGAAGC *********
GTTCAACGACTAAT GTTCAACGACTATA GTTCAACGACTATA GTTCAACGACTAAA ************
AATACATAGTCT AAGAAATAGTCT AAGAAATAGTCT ATGAAATAGTCT * * *******
GATAACAAATTG GATAACAAGTTG GATAACAAGTTG TAACATAAATTG * * ** ***
P4
P6 P7
not only that the insert is co-transcribed with conserved LSU rRNA sequences into a low-abundance, 8-kb precursor RNA, but also that the presumptive intronic segment is spliced from this precursor and retained as a relatively stable 4.4-kb RNA species in the mitochondria of the fungus (Bell et al. 1996). We have sequenced the PCR product that corresponds to the insert in the U11 region of the mitochondrial LSU rRNA gene of C. parasitica strain Ep155 and found that it is a 4238-bp segment of DNA that is not part of the highly conserved nucleotide sequences of the mature mitochondrial large-subunit rRNAs. As shown in Fig. 1 and Table 1, this nucleotide sequence has features characteristic of group-I introns, including a U at the 3′ end of the 5′ exon, a G at the 3′ end of the intron, an internal guide sequence (IGS) adjacent to the 5′ splicing site, and the P, Q, R, S structural elements involved in the formation of the catalytic core of “self-splicing” elements of this type (Waring and Davies 1984; Burke et al. 1987; Cech 1988, 1990). This group-I intron might be the largest of its kind detected so far in a fungus, for the longest that has been described previously is the 2631-bp ND1i2 intron in the mtDNA of P. anserina (Cummings et al.1990 a, 1990 b). Translation of the intronic DNA in all six reading frames revealed only two ORFs encoding polypetides longer than 80 amino acids. The first ORF, called ORF-rpm because it appears to encode a ribosomal protein (RP) and a maturase (M), begins with an ATG initiator codon at position 1221 of the nucleotide sequence shown in Fig. 1 (nucleotide 1168 of the intron), and ends at position 3773, where it is followed by the most common mitochondrial terminator codon, TAA. Thus, ORF-rpm is entirely contained within the intron and potentially encodes a polypeptide chain of 851 amino acids, which appears to be the longest mitochondrial intronic protein found to date. The second ORF, called ORF-tm because it appears to encode a truncated maturase (TM), starts at position 3859 of the nucleotide sequence shown in Fig. 1, lacks a typical mitochondrial translation- initiation codon, and ends at position 4107, where it is followed by the TAG terminator codon. A BLAST search of sequence databases using the predicted 851 amino-acid sequence encoded by ORF-rpm in-
dicated a high degree of similarity with two types of proteins encoded by intronic ORFs in fungal mtDNAs. As shown in Fig. 2, a block of 510 amino acids (residues 31–540 in Fig. 1) from the amino-terminal portion of the putative protein has a sequence similar to the amino-acid sequences of a group of closely related proteins encoded by ORFs located in introns situated in the U11 region of the mitochondrial rRNAs of P. anserina (Cummings et al. 1989 a), N. crassa (Burke and RajBhandary 1982), Penicillium chrysogenum (Naruse et al. 1993) and Aspergillus nidulans (Netzker et al. 1982). In N. crassa, this polypeptide has been identified as the mitochondrial small subunit ribosomal protein, S5 (LaPolla and Lambowitz 1981; Burke and RajBhandary 1982), and it is assumed that related proteins of the other fungi have similar functions. The similarity between all the putative S5 proteins appears to start at the respective amino termini, except that this region of the C. parasitica polypeptide is expanded by an insertion of about 50 amino acids. This observation leads to two options which have not been resolved at this time: (1) the S5 polypeptide of C. parasitica is extended relative to the S5 proteins of other fungal species because an insertion of about 150 nucleotides has occurred somewhere in the region that is located immediately downstream from the initiator codon at position 1221, or (2) translation of the RPM polypeptide is initiated at the second AUG within ORF-rpm (position 1311 in Fig. 1) on the intronic transcript such that the resulting polypeptide is only a few amino acids longer than the related proteins. As shown in Fig. 3, the carboxyl-terminal 311 amino acids (residues 541–851 inclusive) of the putative RPM polypeptide are arranged in a sequence that is similar to those of the putative proteins encoded by ORFs found in the following mitochondrial group-I introns: COIi5, ND5i2 and ND3i1 of P. anserina (Cummings et al. 1989 b), ND5i1 of N. crassa (Nelson and Macino 1987), and the one located in the SSU rRNA gene in the mtDNA of Sclerotinia sclerotiorum (Carbone et al. 1995). Proteins encoded by these ORFs are identified as “maturases” because they contain two well-conserved copies of the LAGLI-DADG dodecapeptide sequence motif and a sec-
112 Fig. 1 Nucleotide sequence of the 4238-bp, group-I intron in the U11 region of the mitochondrial LSU rRNA gene of C. parasitica and the amino-acid sequences of the putative proteins encoded by open reading frames on the sense strand of the DNA. The exon/intron junctions have been determined based on sequence homology. The flanking exon sequences were obtained by DNA sequence analysis of C. parasitica mtDNA HindIII clones determined to contain the segments of the 3′ end of the LSU rRNA gene. Conserved sequence elements (IGS, P, Q, R and S) are underlined and labeled above the nucleotide sequence. Pertinent amino- and carboxyl-termini, as well as dodecapeptide and rA and rB motifs, are labeled below the amino-acid sequences of the corresponding putative proteins. Possible translation initiation and termination codons, as well as a putative Shine-Delgarno (S-D) sequence (AGGAG) which ends 10 bp upstream of the first of the two potential initiator codons for ORF-rpm, have been highlighted as bold capital letters in the nucleotide sequence. The first 30 amino acids of the putative S5 protein are shown in italics because similar sequences are non-extant in the S5-like mitochondrial proteins of other fungi. The nucleotide sequence of the intron and the flanking U11 regions of the LSU rRNA can be found under accession number AF068139 in the GenBank sequence data library
ond set of repeated sequences referred to as r A and rB (Cummings et al. 1989 b). The r A and rB motifs are located at the same distance downstream from the first and second dodecapeptide, respectively. These ORFs also show evidence of having expanded by a duplication event because the segments that are located downstream from each
dodecapeptide are very similar to each other in amino-acid sequence and length. The above observations indicate that the long ORF-rpm encodes a polypeptide that has arisen by fusion of a complete S5 ribosomal protein to an entire maturase polypeptide. Whether or not a functional S5 ribosomal protein and
113 Fig. 2 Comparison of the deduced amino-acid sequence of the putative S5 ribosomal protein encoded by the 5′ terminal portion of the intronic ORFrpm of C. parasitica (C. p.) with the sequences of similar proteins encoded by ORFs found in introns located in the U11 region of the mitochondrial LSU rRNA of P. anserina (P. a.), N. crassa (N. c.), P. chrysogenum (P. c.), and A. nidulans (A. n.). Because of an error at a position between nucleotides 1908 and 1917 in the nucleotide sequence of the N. crassa intron, the sequence of the 180 amino acids located proximal to the carboxyl terminus of the corresponding S5 protein shown in this figure differs from that which was published previously (Burke and RajBhandary 1982). Amino acids that are identical in all five proteins are indicated by asterisks (*), whereas positions occupied by amino-acids of similar characteristics are indicated by dots (·)
an active maturase or homing DNA endonuclease are actually generated in the mitochondria, either by processing of the putative RPM precursor protein or by selective translation of the intronic RNA, remains to be demonstrated. In this context, it may be worth noting that an ATG that could be the initiation codon for the maturase begins at position 2827 of the nucleotide sequence shown in Fig. 1, only five codons upstream of the tyrosine that appears to be the amino-terminal amino acid of this protein. The significance of this observation, however, is debatable because the methionine encoded by this particular ATG is located in a highly conserved domain of the S5 polypeptide and represents a conservative substitution for the valine residues that are located at the same position of the other four fungal mitochondrial S5 proteins (see Fig. 2). Hence, it is more likely that the putative S5 ribosomal protein and the maturase are initially co-translated and then generated post-translationally either by an autocatalytic cleavage or through processing by a specific protease. Indeed, there are several examples of inteins (protein splicing introns; Colston and Davis 1994), some of which are proteins with dodecapeptide motifs similar to those found in maturases (Shub and Goodrich-Blair 1992; Dalgaard et al. 1997).
Maturases and maturase-like proteins constitute the majority of mitochondrial intron-encoded polypeptides in fungi. Whereas most of these proteins seem to promote RNA splicing, some maturases with the LAGLI-DADG motifs cleave DNA. Intron-encoded maturase-like proteins that cleave DNA appear to be involved primarily in intron homing (Lambowitz 1989; Lambowitz and Belfort 1993). In Saccharomyces pombe, the first intron of cox1 encodes a maturase which appears to be involved in intron (RNA) splicing, as well as intron homing (Schäfer et al. 1994). Similar activities have been surmised for the proteins encoded by the ORFs in the aI4α intron in the COX1 gene of Saccharomyces cerevisiae (Wenzlau et al. 1989; Henke et al. 1995) and the intron in the apocytochrome b gene of A. nidulans (Ho et al. 1997). Furthermore, it has been demonstrated that modification of the amino-acid sequence can turn RNA maturases into DNA endonucleases (Labouesse et al. 1987; Goguel et al. 1992; Pel and Grivell 1993; Szczepanek and Lazowska 1996), suggesting an evolutionary link between endonuclease and maturase activity (Lambowitz and Belfort 1993). Cummings et al. (1989 b) speculated that the rA and rB elements of maturases found in the P. anserina CO1i5 and related introns may be symp-
114 Fig. 3 Comparison of the deduced amino-acid sequence of the putative maturase (C. p. LSU) encoded by the 3′ terminal portion of ORF-rpm in the C. parasitica mitochondrial LSU rRNA gene intron with similar proteins encoded by ORFs in the ND3i1, ND4Li1, ND5i2, and CO1i5 introns of P. anserina (Cummings et al. 1989 b). Amino acids that are identical in all five proteins are indicated by asterisks (*), positions occupied by amino-acids of similar characteristics are indicated by dots (·)
*
tomatic of DNA-specific endonuclease activity. On the basis of these considerations, it is tempting to suggest that the maturase encoded by the C. parasitica mitochondrial LSU rRNA gene was derived by transposition from an intron capable of homing. This concept is supported by the observation that the N. crassa mitochondrial LSU rRNA gene U11 intron does encode an S5 protein, but does not encode a maturase. This particular intron appears to be selfsplicing in vitro (Garriga and Lambowitz 1986), whereas splicing in vivo is dependent on ancillary proteins encoded by nuclear genes believed to be required for proper folding of the unprocessed precursor transcript (Akins and Lambowitz 1987; Lambowitz and Perlman 1990; Caprara et al. 1996). Hence, the acquired maturase-like protein may be redundant for efficient splicing of the LSU rRNA precursor in C. parasitica. There is also no basis for assuming that the acquired maturase-like protein has been seconded as an endonuclease for homing and/or a factor involved in the cleavage of the putative RPM polypeptide because it has not been shown that the corresponding intron is mobile or that the rpm ORF is translated into a product that is subsequently processed to yield S5 and maturase-like proteins. One of the unique features of the C. parasitica intron is that the portion of ORF-rpm that encodes the maturaselike protein is fused in-frame to another intronic ORF, this one encoding a ribosomal protein. While many group-I intronic ORFs, particularly those encoding maturase-like proteins, are continuous with unrelated open reading frames, they usually are fused to upstream exons (Grivell 1995), and there is some evidence indicating that the resulting exon-intron fusion proteins are processed to release
the intron-encoded proteins (Weiss-Brummer et al. 1982; reviewed by Lambowitz and Belfort 1993; Pel and Grivell 1993; Grivell 1995). The presence of two ORFs in one intron has also been previously documented (Cummings et al. 1988, 1989 b), but in these cases both ORFs encode maturases and the first is continuous with a short 5′ exon, whereas the second is disconnected from the upstream ORF as well as from exonic coding segments. A higher degree of autonomy exists in the cellular slime mold Dictyostelium discoideum, where neither of the two related freestanding ORFs that have been found in an intron in the gene coding for the fusion product of cytochrome oxidase subunits 1 and 2 is connected to an exonic reading frame (Ogawa et al. 1997). In contrast, ORF-rpm is not only the first reported case of a fusion of two intronic ORFs, but also of the presence in the same intron of a gene encoding the S5 ribosomal protein together with an ORF that encodes a maturase-like protein. In P. anserina (Cummings et al. 1989 a) and Ophiostoma novo-ulmi (Abu-Amero et al. 1995), the mitochondrial LSU rRNA gene contains two group-I introns, of which the first one encodes a maturase, whereas the second one, which is located within the U11 region, encodes a putative S5 protein. In contrast, the same gene of N. crassa contains only one group-I intron, which is located in the U11 region and contains a freestanding ORF that encodes the S5 protein (Burke and RajBhandary 1982). Thus, there is considerable variation with respect to the arrangement of intronic ORFs even amongst fungi which belong to the same monophyletic group on the basis of rRNA sequence data (reviewed in Alexopoulos et al. 1996; Chen et al. 1996). In this respect, the arrangement of the putative S5 and maturase genes
115
Fig. 4 Comparison of the deduced amino-acid sequence of the truncated maturase protein (C. p. TM) encoded by a segment of DNA proximal to the 3′ end of the intron in the LSU rRNA gene of C. parasitica with segments of the amino-acid sequences of the putative maturases encoded by: (1) the intronic ORF-rpm of the same organism (C. p. RPM), (2) an ORF in the intron within ND4L of N. crassa (N. c. ND4L), (3) an ORF in the second intron of P. anserina ND4L (P. a. ND4Li2), (4) a truncated short ORF found in the second intron of the P. anserina LSU rRNA (P. a. r2; Cummings et al. 1989 a), and (5) an open reading frame in the intron of the N. crassa mitochondrial gene encoding subunit 6 of the mitochondrial ATPase (N. c. ATPase6i). Asterisks (*) indicate the positions in all five proteins occupied by the same amino acid, dots (·) indicate positions occupied by functionally related amino acids in all five proteins, and dashes (–) indicate that the amino acid found at this position in the TM sequence is also present in at least two other sequences in the alignment
within the intron in C. parasitica further supports the current view that the structural and coding regions of group-I introns evolve independently of each other (Cummings and Domenico 1988; Mota and Collins 1988; Lambowitz and Belfort 1993), possibly because the ORFs themselves are mobile genetic elements (Loizos et al. 1994; Sellem et al. 1996; Sellem and Belcour 1997). It has been suggested that the fusion of maturase-encoding intronic ORFs to upstream exons provides a feedback mechanism such that slower splicing results in the production of more maturase, which in turn accelerates splicing and the production of the protein encoded by the exons of the host gene (Lazowska et al. 1980; Ohta et al. 1993). In contrast, the intron-encoded maturase-like polypeptide of C. parasitica is fused to the S5 ORF, which is totally disconnected from an exonic coding region. Thus, there is no obvious way by which the splicing of the primary transcript of the LSU rRNA might affect the synthesis of the maturase-like protein, or vice versa. Furthermore, it is not apparent how the maturase might co-ordinate the synthesis of a small subunit ribosomal protein, S5, with the synthesis of the LSU rRNA without assuming that the cleavage of the intron from the primary transcript affects the rate of translation of ORF-rpm and/or the generation of S5 and the maturase-like protein from the RPM precursor polypeptide. The presence of more than one ORF in one intron has been viewed as support for the possibility that intronic genes by themselves are mobile genetic elements (Sellem et al. 1996; Sellem and Belcour 1997). This raises some interesting questions regarding the origin of the long and mosaic ORF in the C. parasitica mitochondrial LSU rRNA intron. A clue may be provided by the presence of the 83-codon, truncated maturase reading frame proximal to the 3′ end of the intron. A segment of this reading frame
shares a high degree of sequence similarity with a small truncated ORF found in the intron located in the U11 region of the mitochondrial LSU rRNA gene of P. anserina (Fig. 4; Cummings et al. 1989 a). A part of each of these truncated ORFs is similar to a segment of the complete maturases encoded by other mitochondrial introns, particularly ND4Li2 of P. anserina (Cummings et al. 1990 a) and the introns in ND4L (Nelson and Macino 1987) and ATPase6 (Morelli and Macino 1984) of N. crassa. The similarity starts at the end of the first dodecapeptide and continues for about 42 amino acids. This partial ORF could be the end product of a recombination event, the remnant of a failed ORF homing event, or the result of an incomplete excision event. In any case, the presence of ORF-tm in the intron that is located in the U11 region of C. parasitica suggests that the corresponding intron in the mtDNA of an ancestral species may have been similar to that which persists in P. anserina. If so, then the ORF-rpm of C. parasitica is the product of a transposition or an erroneous homing event by which a nucleotide sequence encoding a maturase-like protein, and possibly a stretch of nucleotides located downstream from the coding region, was added to the resident S5 ORF. The fact that the inserted ORF encodes an apparently complete maturase-like protein suggests that it might have originated by reverse transcription from the 5′ end of an intronic RNA encoding a maturase that is fused in-frame to an upstream exon. Although highly variable in sequence, group-I introns usually occur in highly conservative regions and/or highly conserved nucleotide sequences of mitochondrial genes. In this respect, the long intron of C. parasitica is no exception, for it is located in the U11 region near the 3′ end of the mitochondrial LSU RRNA gene. Group-I introns varying in length from 1132 to 2404 bp are located at equivalent sites in the mitochondrial LSU rRNA genes of species as diverse as the yeasts S. cerevisiae, Kluyveromyces spp. and Hansenula wingei (Dujon 1980; Jacquier and Dujon 1983; Sekito et al. 1995), and the filamentous fungi N. crassa, P. anserina, P. chrysogenum and A. nidulans (Burke and RajBhandary 1982; Cummings et al. 1989 a, Naruse et al. 1993). While unusual because of its size, the 4.3-kb intron appears to be a distinguishing feature of most, if not all strains of C. parasitica: based on PCR analysis with the IP1 and IP2 primers, a 4.3-kb PCR product could be generated from the genomic DNAs from several strains of diverse geographical origins in North America and Europe, the majority being contained in the collection that was used by Bell et al. (1996) to assess diversity in the mitochondrial genome of this organism (data not shown).
116
Mitochondrial group-I introns might be useful targets for the induction of cytoplasmically transmissible forms of hypovirulence in pathogenic strains of C. parasitica and other fungi. A cytoplasmically transmissible hypovirulence phenotype has been associated with respiration deficiency and mitochondrial defects in C. parasitica (Monteiro-Vitorello et al. 1995). Indeed, plasmid-like mitochondrial derivatives similar to the senDNAs of Podospora have been observed in diseased, hypovirulent isolates of O. novo-ulmi (Abu-Amero et al. 1995) and C. parasitica (Monteiro-Vitorello et al. 1995 and unpublished results). In both cases, these elements appear to have arisen by rearrangements involving intronic sequences located in mitochondrial rRNA genes. On the basis of these observations, it is likely that at least some of the abnormal mtDNA derivatives that result from abnormal splicing (Dujon 1989) and recombination events in intronic nucleotide sequences (Dujon and Belcour 1989; Jamet-Vierny et al. 1997) produce mutants that have contagious hypovirulence phenotypes that are associated with respiratory defects. Therefore, the elucidation of the structure and biology of the group-I introns in C. parasitica, the paradigm of the wound-infecting phytopathogenic fungi, is valuable for the understanding of mitochondrial hypovirulence syndromes and their use in the biological control of this group of organisms. Acknowledgements We thank Dr. Dennis W. Fulbright for providing the C. parasitica strain used in this study and Katherine A. Nummy for technical assistance. This work was supported by the U.S. Department of Agriculture National Research Initiative Competitive Grants Program (95-37303-1785). G. H. was the recipient of a Postdoctoral Fellowship from the Natural Science and Engineering Research Council of Canada.
References Abu-Amero SN, Charter NW, Buck KW, Brasier CM (1995) Nucleotide-sequence analysis indicates that a DNA plasmid in a diseased isolate of Ophiostoma novo-ulmi is derived by recombination between two long repeat sequences in the mitochondrial large subunit ribosomal RNA gene. Curr Genet 28: 54–59 Akins RA, Lambowitz AM (1987) A protein required for splicing group-I introns in Neurospora mitochondria is mitochondrial tyrosyl-tRNA synthetase or a derivative thereof. Cell 50: 331–345 Alexopoulos CJ, Mims CW, Blackwell M (1996) Introductory mycology, 4th edn. John Wiley and Sons Inc, New York Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic logical alignment search tool. J Mol Biol 215: 403–410 Belcour L, Rossignol M, Koll F, Sellem CH, Oldani C (1997) Plasticity of the mitochondrial genome in Podospora. Polymorphisms for 15 optional sequences: group-I, group-II introns, intronic ORFs and an intergenic region. Curr Genet 31: 308–317 Bell JA, Monteiro-Vitorello CB, Hausner G, Fulbright DW, Bertrand H (1996) Physical and genetic map of the mitochondrial genome of Cryphonectria parasitica Ep155. Curr Genet 30: 34–43. Burke JM, RajBhandary UL (1982) Intron within the large rRNA gene of N. crassa mitochondria: a long open reading frame and a consensus sequence possibly important in splicing. Cell 31: 509–520 Burke JM, Belfort M, Cech TR, Davies RW, Schweyen RJ, Shub DA, Szostak JW, Tabak HF (1987) Structural conventions for groupI introns. Nucleic Acids Res 15: 7217–7222
Caprara MG, Mohr G, Lambowitz AM (1996) A tyrosyl tRNA synthetase protein induces tertiary folding of the group-I intron catalytic core. J Mol Biol 257: 512–531 Carbone I, Anderson JB, Kohn LM (1995) A group-I intron in the mitochondrial small subunit ribosomal RNA gene of Sclerotinia sclerotiorum. Curr Genet 27: 166–176 Cech TR (1988) Conserved sequences and structures of group-I introns: building an active site for RNA catalysis – a review. Gene 73: 259–271 Cech TR (1990) Self-splicing of group-I introns. Annu Rev Biochem 55: 599–629 Chen B, Chen C-H, Bowman BH, Nuss DL (1996) Phenotypic changes associated with wild-type and mutant hypovirus RNA transfection of plant pathogenic fungi phylogenetically related to Cryphonectria parasitica. Phytopathology 86: 301–310 Collins RA (1993) Neurospora crassa laboratory strain 74-OR231A: mitochondrial genes. In: O’Brien SJ (ed) Genetic maps, 6th edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, pp 3.19–3.21 Colston MJ, Davis EO (1994) The ins and outs of protein splicing elements. Mol Microbiol 12: 359–363 Cummings DJ, Domenico J (1988) Sequence analysis of mitochondrial DNA from Podospora anserina. J Mol Biol 204: 815–839 Cummings DJ, Domenico JM, Michel F (1988) DNA sequence and organization of the mitochondrial ND1 gene from Podospora anserina: analysis of alternate splice sites. Curr Genet 14: 253–264 Cummings DJ, Domenico JM, Nelson J (1989 a) DNA sequence and secondary structures of the large subunit rRNA coding regions and its two class-I introns of mitochondrial DNA from Podospora anserina. J Mol Evol 28: 242–255 Cummings DJ, Michel F, McNally KL (1989 b) DNA sequence analysis of the 24.5-kilobase pair cytochrome oxidase subunit I mitochondrial gene from Podospora anserina: a gene with 16 introns. Curr Genet 16: 381–406 Cummings DJ, Michel F, Domenico JM, McNally KL (1990 a) DNA sequence analysis of the mitochondrial ND4L-ND5 complex from Podospora anserina. J Mol Biol 212: 269–286 Cummings DJ, McNally KL, Domenico JM, Matsuura ET (1990 b) The complete DNA sequence of the mitochondrial genome of Podospora anserina. Curr Genet 17: 375–402 Dalgaard JZ, Klar AJ, Moser MJ, Holley WR, Chatterjee A, Mian IS (1997) Statistical modeling and analysis of the LAGLIDADG family of site-specific endonucleases and identification of an intein that encodes a site-specific endonuclease of the HNH family. Nucleic Acids Res 25: 4626–4638 Dujon B (1980) Sequence of the intron and flanking exons of the mitochondrial 21 S rRNA gene of yeast strains having different alleles at the omega and rib-1 locus. Cell 20: 185–197 Dujon B (1989) Group-I introns as mobile genetic elements: facts and mechanistic speculation – a review. Gene 82: 91–114 Dujon B, Belcour L (1989) Mitochondrial DNA instabilities and rearrangements in yeasts and fungi. In: Mobile DNA Berg DE, Howe MM (eds), American Society of Microbiology, Washington D.C., pp 861–878 Garriga G, Lambowitz AM (1986) Protein-dependent splicing of a group-I intron in ribonucleoprotein particles and soluble fractions. Cell 46: 669–680 Gillham NM (1994) Organelle genes and genomes. Oxford University Press, New York Goguel V, Delahodde A, Jacq C (1992) Connections between RNA splicing and DNA intron mobility in yeast mitochondria: RNA maturase and DNA endonuclease switching experiments. Mol Cell Biol 12: 696–705 Grivell LA (1995) Nucleo-mitochondrial interactions in mitochondrial gene expression. Crit Rev Biochem Mol Biol 30: 121–164 Gutell RR, Fox GE (1988) A compilation of large subunit rRNA sequences represented in a structural format. Nucleic Acids Res 16: r175-r269 Henke MR, Butow RA, Perlman PS (1995) Maturase and endonuclease functions depend on separate conserved domains of the bifunctional protein encoded by the group-I intron aI4alpha of yeast mitochondrial DNA. EMBO J 14: 5094–5099
117 Hensgens LAM, Bonen L, De Haan M, Van der Horst G, Grivell LA (1983) Two intron sequences in the yeast mitochondrial COXI gene: homology among URF-containing introns and strain-dependent variation in flanking exons. Cell 32: 379–389 Ho Y, Kim S-J, Waring RB (1997) A protein encoded by a group-I intron in Aspergillus nidulans directly assists RNA splicing and is a DNA endonuclease. Proc Natl Acad Sci USA 94: 9894–8999 Jacquier A, Dujon B (1983) The intron of the mitochondrial 21S rRNA gene: distribution in different yeast species and sequence comparison between Kluyveromyces thermotolerans and Saccharomyces cerevisiae. Mol Gen Genet 192: 487–499 Jamet-Vierny C, Boulay J, Briand J-F (1997) Intramolecular crossovers generate deleted mitochondrial DNA molecules in Podospora anserina. Curr Genet 31: 162–170 Labouesse M, Herbert, CJ, Dujardin, G, Slonimski, PP (1987) Three suppressor mutations which cure a mitochondrial RNA maturase deficiency occur at the same codon in the open reading frame of the nuclear NAM2 gene. EMBO J 67: 13–21. Lambowitz AM (1989) Infectious introns. Cell 56: 323–326 Lambowitz AM, Belfort M (1993) Introns as mobile genetic elements. Annu Rev Biochem 62: 587–622 Lambowitz AM, Perlman PS (1990) Involvement of aminoacyltRNA synthetases and other proteins in group-I and group-II intron splicing. Trends Biochem Sci 15: 440–444 Lazowska J, Jacq C, Slonimski P (1980) Sequence of introns and flanking exons in wild-type and box3 mutants of cytochrome b reveals an interlaced splicing protein coded by an intron. Cell 22: 333–348 LaPolla RJ, Lambowitz AM (1981) Mitochondrial ribosome assembly in Neurospora crassa. J Biol Chem 256: 7064–7067 Loizos N, Tillier ERM, Belfort M (1994) Evolution of mobile groupI introns: recognition of intron sequences by an intron-encoded endonuclease. Proc Natl Acad Sci USA 91: 11983–11987 Mahanti N, Bertrand H, Monteiro-Vitorello C, Fulbright DW (1993) Elevated mitochondrial alternative oxidase activity in dsRNAfree, hypovirulent isolates of Cryphonectria parasitica. Physiol Mol Plant Pathol 42: 455–463 Michel F, Cummings DJ (1985) Analysis of class-I introns in a mitochondrial plasmid associated with senescence of Podospora anserina reveals extraordinary resemblance to the Tetrahymena ribosomal intron. Curr Genet 10: 69–79 Mishra NC (1991) Genetics and molecular biology of Neurospora crassa. Adv Gen 29: 1–62 Monteiro-Vitorello CB, Bell JA, Fulbright DW, Bertrand H (1995) A cytoplasmically transmissible hypovirulence phenotype associated with mitochondrial DNA mutations in the chestnut blight fungus Cryphonectria parasitica. Proc Natl Acad Sci USA 92: 5935–5939 Morelli G, Macino G (1984) Two intervening sequences in the ATPase subunit 6 of Neurospora crassa. A short intron (93 base pairs) and a long intron that is stable after excision. J Mol Biol 178: 491–507 Mota EM, Collins RA (1988) Independent evolution of structural and coding regions in a Neurospora mitochondrial intron. Nature 332: 654–656 Naruse A, Yamamoto H, Sekiguchi J (1993) Nucleotide sequence of the large mitochondrial rRNA gene of Penicillium chrysogenum. Biochim Biophys Acta 1172: 353–356 Nelson MA, Macino G (1987) Three class-I introns in the ND4L/ND5 transcriptional unit of Neurospora crassa mitochondria. Mol Gen Genet 206: 318–325 Netzker R, Kochel HG, Basak N, Küntzel H (1982) Nucleotide sequence of Aspergillus nidulans mitochondrial genes coding for ATPase subunit 6, cytochrome oxidase subunit 3, seven unidentified proteins, four tRNAs and L-rRNA. Nucleic Acids Res 10: 4783–4794
Ogawa S, Matsuo K, Angata K, Yanagisawa K, Tanaka Y (1997) Group-I introns in the cytochrome c oxidase genes of Dictyostelium discoideum: two related ORFs in one loop of a group-I intron, a cox 1/2 hybrid gene and an unusually large cox3 gene. Curr Genet 31: 80–88 Ohta E, Oda K, Yamato K, Nakamura Y, Takemura M, Nozato N, Akashi K, Ohyama K, Michel F (1993) Group-I introns in the liverwort mitochondrial genome: the gene coding for subunit 1 of cytochrome oxidase shares five intron positions with its fungal counterparts. Nucleic Acids Res 21: 1297–1305 Pel HJ, Grivell LA (1993) The biology of yeast mitochondrial introns. Mol Biol Rep 18: 1–13 Rogers HJ, Buck KW, Brasier CM (1987) A mitochondrial target for double–stranded RNA in diseased isolates of the fungus that causes Dutch elm disease. Nature 329: 558–560. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Schäfer B, Wilde B, Massardo R, Manna F, Del-Giudice L, Wolf K (1994) A mitochondrial group-I intron in fission yeast encodes a maturase and is mobile in crosses. Curr Genet 25: 336–341 Sekito T, Okamoto K, Kitano H, Yoshida K (1995) The complete mitochondrial DNA sequence of Hansenula wingei reveals new characteristics of yeast mitochondria. Curr Genet 28: 39–53 Sellem CH, Belcour L (1997) Intron open reading frames as mobile elements and evolution of a group-I intron. Mol Biol Evol 14: 518–526 Sellem CH, d’Aubenton-Carafa Y, Rossignol M, Belcour L (1996) Mitochondrial intronic open reading frames in Podospora: mobility and consecutive exonic sequence variations. Genetics 143: 777–788 Shnyreva AV (1995) Mitochondrial introns in fungi and their evolutionary role. Russian J Gen 31: 741–747 Shub DA, Goodrich-Blair H (1992) Protein introns: a new home of endonucleases. Cell 71: 183–186 Szczepanek T, Lazowska J (1996) Replacement of two non-adjacent amino acids in the S. cerevisiae bi2 intron-encoded RNA maturase is sufficient to gain a homing-endonuclease activity. EMBO J 15: 3758–3767 Weiss-Brummer B, Rodel G, Schweyen RJ, Kaudewitz R (1982) Expression of the split gene cob in yeast: evidence for a precursor of a “maturase” protein translated from intron 4 and preceding exons. Cell 29: 527–536 Waring RB, Davies RW (1984) Assessment of a model for intron RNA secondary structure relevant to RNA self-splicing – a review. Gene 28: 277–291 Wenzlau JM, Saldanha RJ, Butow RA, Perlman PS (1989) A latent intron-encoded maturase is also an endonuclease needed for intron mobility. Cell 56: 421–430 Wolf K, Del Giudice L (1988) The variable mitochondrial genome of ascomycetes: organization, mutational alterations, and expression. Adv Gen 25: 185–308 Yang J, Mohr G, Perlman PS, Lambowitz AM (1998) Group-II intron mobility in yeast mitochondria: target DNA-primed reverse transcription activity of aI1 and reverse splicing into DNA transposition sites in vitro. J Mol Biol 282: 505–523 Zhang W, Hu G, Deisseroth A (1991) Improvement of PCR sequencing by formamide. Nucleic Acids Res 19: 6649
