Identification of the Genes Encoding the Cytosolic Translation Release Factors from Podospora anserina and Analysis of Their Role During the Life Cycle

Corresponding author: Philippe Silar, Institut de Génétique et Microbiologie, URA 2225, Université de Paris-Sud, 91405 Orsay cedex, France., silar{at}igmors.u-psud.fr (E-mail).

Communicating editor: A. G. HINNEBUSCH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In an attempt to decipher their role in the life history and senescence process of the filamentous fungus Podospora anserina, we have cloned the su1 and su2 genes, previously identified as implicated in cytosolic translation fidelity. We show that these genes are the equivalents of the SUP35 and SUP45 genes of Saccharomyces cerevisiae, which encode the cytosolic translation termination factors eRF3 and eRF1, respectively. Mutations in these genes that suppress nonsense mutations may lead to drastic mycelium morphology changes and sexual impairment but have little effect on life span. Deletion of su1, coding for the P. anserina eRF3, is lethal. Diminution of its expression leads to a nonsense suppressor phenotype whereas its overexpression leads to an antisuppressor phenotype. P. anserina eRF3 presents an N-terminal region structurally related to the yeast eRF3 one. Deletion of the N-terminal region of P. anserina eRF3 does not cause any vegetative alteration; especially life span is not changed. However, it promotes a reproductive impairment. Contrary to what happens in S. cerevisiae, deletion of the N terminus of the protein promotes a nonsense suppressor phenotype. Genetic analysis suggests that this domain of eRF3 acts in P. anserina as a cis-activator of the C-terminal portion and is required for proper reproduction.

IN eukaryotes, polypeptide release from the ribosome during the termination step of the cytosolic translation process is mediated by a unique factor called eRF (GOLDSTEIN et al. 1970 ; KONECKI et al. 1977 ). This factor is composed of two subunits, eRF1 and eRF3 (FROLOVA et al. 1994 ; STANSFIELD et al. 1995 ; ZHOURAVLEVA et al. 1995 ). eRF1 possesses the catalytic release activity (FROLOVA et al. 1994 ), whereas eRF3 promotes a GTP-dependent activation of eRF1 (ZHOURAVLEVA et al. 1995 ). The genes encoding eRF1 (SUP45) and eRF3 (SUP35) were genetically identified in the yeast Saccharomyces cerevisiae long ago in screens for recessive mutations that resulted in an increased readthrough at all three stop codons (informational omnipotent suppressors; HAWTHORNE and MORTIMER 1968 ; INGE-VECHTOMOV and ANDRIANOVA 1970 ). Suprisingly, mutations in SUP35 can also promote ribosomal frameshifting (WILSON and CULBERTSON 1988 ). Apart from these direct effects on translation, mutations in these genes entail various physiological alterations whose causes are not obvious (INGE-VECHTOMOV et al. 1994 for review; MIRONOVA et al. 1995 ; TIKHOMIROVA and INGE-VECHTOMOV 1996 ).

Recently, eRF3 has received much attention because it is responsible for the phenomenon in S. cerevisiae. [⁺] strains have a higher level of suppressor tRNA mediated readthrough compared to the [^-] strains. This difference is caused by a non-Mendelian genetic element () that has been shown to be cytoplasmic and infectious (COX 1965 ). It was shown that a mutation located in the SUP35 gene (the PNM2^- mutation; YOUNG and COX 1971 ; DOEL et al. 1994 ) as well as the deletion of the amino terminus of the protein (TER-AVANESYAN et al. 1994 ) eliminate , showing that SUP35 is involved in the generation of . Recently, it was found that in [⁺] strains, the eRF3 protein (and the eRF1 protein bound to it) is aggregated, whereas in [^-] strains it is present at lower amounts in a nonaggregated form (PATINO et al. 1996 ; PAUSHKIN et al. 1996 , PAUSHKIN et al. 1997A ). The passage to the aggregated form of the protein is promoted by conformational changes of the eRF3 protein, related to the ones that are thought to be involved in the neurodegenerative diseases caused by prions (WICKNER 1994 ; GLOVER et al. 1997 ; KING et al. 1997 ; PAUSHKIN et al. 1997B ). These changes are mediated by the amino-terminal portion of the protein, which is dispensable for the eRF1 GTP-dependent activation (ZHOURAVLEVA et al. 1995 ; DERKATCH et al. 1996 ; GLOVER et al. 1997 ). Deletion of this portion of eRF3 results in decreased readthrough at stop codon (antisuppression), suggesting that this part of the protein acts as a cis-repressor of the carboxy terminus of the protein (TER-AVANESYAN et al. 1993 ). In yeast, this N-terminal deletion does not seem to be associated with other phenotypes.

It has recently been proposed that this phenomenon is a novel strategy enabling yeast colonies to cope with new environmental conditions (LINDQUIST 1997 ). It is not known whether the eRF3 proteins of the other organisms present the potentiality in vivo or in vitro to adopt different structures. However, like the yeast protein, the eRF3 proteins known in diverse eukaryotes all seem to contain two domains which have been conserved differently during evolution (Figure 2): the carboxy terminus of the protein, involved in translation termination which is very conserved, and an amino-terminal extension with varying length and primary structure.

View larger version (78K): In this window In a new window Download PPT slide   Figure 1. Comparison of P. anserina eRF1 to that of yeast (BRIENING and PIPERSBERG 1986 ) and human (GRENETT et al. 1992 ).

View larger version (157K): In this window In a new window Download PPT slide   Figure 2. Comparison of P. anserina eRF3 to the other known eRF3 proteins from Saccharomyces cerevisiae (KUSHNIROV et al. 1988 ), another yeast Pichia pinus (KUSHNIROV et al. 1990 ), and two vertebrates, Xenopus (ZHOURAVLEVA et al. 1995 ) and human (JEAN-JEAN et al. 1996 ). The conserved carboxy terminus starts at position 294 (numbering refers to the P. anserina protein). Asparagine, glutamine, glycine, and tyrosine present in the amino terminus of the fungal proteins are in bold to highlight the richness of this part of the proteins for these amino acids. The repeated sequences present in the P. anserina protein are underlined twice (core repeat) or once (larger repeat).

Podospora anserina is a filamentous fungus easily amenable to molecular genetics. It stands at the borderline between unicellular and multicellular organisms and displays a more complex life history than S. cerevisiae. In addition to the hyphae produced during vegetative growth, it differentiates male (conidia) and female (ascogonia) sexual organs. Fecundation is followed by a morphogenetic process, resulting in the building of a fruiting body (the perithecium) where meioses occur. It also presents an aging process, called "senescence," which limits the proliferative capacity of the cells (RIZET 1953 ). It has been shown that this process is caused by the appearance and subsequent accumulation of a cytoplasmic and infectious factor and that it is associated with a destabilization of the mitochondrial DNA (MARCOU 1961 ; CUMMINGS et al. 1979 ).

Many mutations affecting UGA readthrough have been recovered in this organism and assigned to different genes (PICARD 1973 ; COPPIN-RAYNAL et al. 1988 for a review). Most of them alter several aspects of the life cycle of this organism, especially differentiation of female organs, formation of ascospores or life span. Some of these genes (su4, su8, su12, AS1, and AS4) have already been cloned (DEBUCHY and BRYGOO 1985 ; DEQUARD-CHABLAT and SELLEM 1994 ; SILAR and PICARD 1994 ; SILAR et al. 1997 ), but none so far encodes eRF1 or eRF3. The su1 and su2 genes are good candidates for encoding these factors. Indeed, the many mutations in these genes display the same recessive omnipotent suppressor phenotype as the S. cerevisiae SUP35 and SUP45 mutations (PICARD 1973 ) and it has been shown that the two genes interact (PICARD-BENNOUN 1976 ). We have thus initiated their cloning as well as the analysis of their role in various aspects of P. anserina physiology.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and growth conditions:
All the strains were derived from the P. anserina wild-type S strain (BERNET 1967 ). The su1 and su2 genes were originally identified by screening for mutations that suppress the 193 nonsense spore color mutation (PICARD 1973 ). Spores carrying the 193 mutation are white instead of black (dark green). Presence of a suppressor mutation in su1 or in su2 (as well as in other suppressor loci) restores partial spore pigmentation; i.e., the spores are green. Initially, two types of omnipotent suppressor mutations in the su1 and su2 loci were selected (PICARD 1973 ): (1) "strong suppressors," which, besides a strong effect on translational readthrough, entail important fertility and growth defects, like the su1-25 or su2-5 alleles; (2) "weak suppressors" that display a weaker suppressor activity and give no physiological alterations, like su1-1 or su2-1. These mutations are recessive to wild type. Extragenic mutations, that antagonize various su1 and su2 suppressor mutant alleles, were also screened (antisuppressor mutations). Some of these extragenic mutations map to su1 (for mutations that antagonize su2) and su2 (for mutations that antagonize su1) and display a suppressor phenotype when present alone in the strain (PICARD-BENNOUN 1976 ), suggesting that the proteins encoded by the two genes interact. This is confirmed by a double mutant analysis with other su1 and su2 alleles, which shows that, depending on the su1 and su2 allele combinations, either additive, antagonistic, or equal effect of the mutations is observed (PICARD-BENNOUN 1976 ). In the same study, the AS2 gene is identified as mapping to the same locus as su2 (PICARD-BENNOUN 1976 ). Mutations in this gene antagonize su1 mutations but do not display suppressor phenotype. However, the inability to perform a complementation test between the AS2 and su2 mutations prevents determining whether AS2 mutations are su2 alleles that do not display a suppressor effect on their own or if they belong to another closely linked gene. Table 1 summarizes the main properties of su1 and su2/AS2 mutations.

All the culture conditions and genetic methods used for P. anserina have already been described (ESSER 1974 ). With this organism, monokaryotic or dikaryotic ascopores can be obtained after meiosis, allowing the recovery of either homokaryotic or heterokaryotic strains after crosses. Suppression level was measured as described in PICARD 1973 or COPPIN-RAYNAL 1981 . Measure of longevity was performed as in SILAR and PICARD 1994 . Transformations of protoplasts were done according to BRYGOO and DEBUCHY 1985 . Phenotype of the strains carrying the transgenes was ascertained in at least two successive generations, on at least two independent integrations.

Plasmids and nucleic acids manipulations:
All the DNA and RNA manipulations were performed using standard methods (AUSUBEL et al. 1987 ). mtDNA extraction was done as described in LECELLIER and SILAR 1994 . For the sequence of the su2 cDNA, RNA was extracted from wild type and treated with RNAse-free DNAse I, reverse transcribed and the resulting products were amplified by PCR with the following oligonucleotides: 5'-GAAATCGGCAGAACCAGCAA-3' and 5'-GATTCGCTTCAGTCGTTAAT-3'. The obtained PCR product was then directly sequenced. The oligonucleotides used to amplify the su2 genes in the AS2-1 and AS2-2 mutant strains have the following sequences: 5'-TCCCAGGTTTCTGTATGATA-3' and 5'-ATGCAGTGTCTCCGCTCGG-3'.

Screening for su1 and su2:
The two P. anserina genomic banks used to clone su1 and su2 genes were constructed in the pMoCosX plasmid (ORBACH 1994 ) and were derived from the s strain. S and s differed by two incompatibility genes (BERNET 1967 ). SUP35 and SUP45 DNA fragments used as probes were from plasmids SUP35pEMBLyex4 (a 2.3-kb PstI fragment; TER-AVANESYAN et al. 1993 ; a kind gift from Dr. M. D. TER-AVANESYAN) and pUKC604 (a 1.5-kb XhoI-BglII fragment; a kind gift from Dr. M. F. TUITE), respectively. One clone, named C₂(4), was obtained by screening a first genomic bank with SUP35 probe; however, no positive clone was obtained with the SUP45 one. We thus used a second bank to screen with the SUP45 probe and then obtained two different clones, 16(3) and 6(b). Further analysis revealed that C₂(4) and 16(3) contained the complete homologues of SUP35 and SUP45, respectively.

Subcloning of cosmids C₂(4) and 16(3) was achieved in the pBC-hygro plasmid (SILAR 1995 ; see RESULTS). A 3-kb EcoRI DNA fragment encompassing the totality of su1 was obtained. Subcloning of 16(3) leads to a minimal 5-kb PstI DNA fragment that contains the integrality of the su2 gene. Southern blotting experiments verified that the cloned copies of su1 and su2 were colinear with the genomic copies and that su1 and su2 were unique genes in the P. anserina genome (data not shown).

Deletion of su1:
Based on data obtained in S. cerevisiae, it was most likely that deletion of su1 was lethal. Thus, in order to achieve the deletion, we devised the following scheme: We first constructed a strain carrying the su1-25 allele at the su1 locus and the T4 transgene that is a su1⁺ wild-type copy of the gene, at an ectopic position, tagged with the hygromycin B resistance marker (to ensure survival when the su1-25 allele was deleted). This strain was then transformed with the linearized plasmid "psu1" and phleomycin-resistant transformants were selected. Linearized psu1 was constituted by the complete pBC-phleo plasmid (SILAR 1995 ) inserted between the EcoRI-SacII fragment of su1 (containing the promoter sequences and about 350 bp of the beginning of the su1 coding sequence) and the ApaI-EcoRI fragment (containing the last 500 bp of su1). Upon correct integration at the su1 locus by a double crossing over, this DNA fragment should inactivate the su1-25 allele. This scheme also yielded a screen to eliminate the integrations that occurred elsewhere in the genome (which were the vast majority of the integrations). Indeed, if su1-25 was not inactivated, a cross with a strain carrying the 193 mutation should produce some green spores, whereas only white spores should be observed in inactivation of su1-25. Of the 77 transformants tested, one yielded only white spores when crossed with the strain carrying the 193 mutation. Southern blot analysis confirmed that integration of psu1 did disrupt the su1 locus in this transformant (data not shown). In this system, the deletion is easily followed because of resistance to phleomycin and the wild-type copy is followed because of resistance to hygromycin B. Crossing of this strain with wild type allowed us to retrieve a heterokaryotic strain carrying wild-type and su1-deleted nuclei, free from the wild-type ectopic copy.

Expression of the protein deleted from its N terminus:
To express the P. anserina eRF3 protein truncated for its N terminus, we made a construct containing the promoter region of su1, including the AUG initiator codon, fused in frame with codon leucine number 291. This modified gene was inserted into vector pBC-hygro and the resulting plasmid was transformed in a su1-25 strain. Three transformants were randomly picked for further analysis. The transgenes were subsequently reassociated with their various genetic backgrounds by crosses with appropriate strains (see RESULTS).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The search for extragenic suppressors of the 193 UGA nonsense spore color mutation in P. anserina led to the identification of several loci implicated in translational fidelity (COPPIN-RAYNAL et al. 1988 for review). A majority of these mutations occurred in two genes, su1 and su2. Table 1 gives a description of the principal biological alterations entailed by the su1 and su2 mutations. The mutations may promote alterations of the vegetative growth (growth speed, morphology and longevity of the mycelium) and sexual reproduction (inability to differentiate female organs and alteration of spore germination). Roughly, three classes could be detected: (1) strong suppressors that completely abolish female fertility and that cause severe vegetative growth defects, (2) weak suppressors that do not modify the mycelium physiology and fertility, and (3) intermediate suppressors that lower but do not abolish fertility. Surprisingly, the alteration of longevity is marginal for all classes of mutations except for the su1-50 and su1-51 alleles. MtDNA from senescent cultures was analyzed for some su1 and su2 mutant strains (Table 1). Data indicate that, unlike for the su12 or AS1 genes (BELCOUR et al. 1991 ; SILAR et al. 1997 ), the mtDNA was altered as in wild type during the terminal phase of senescence.

su1 and su2 are the P. anserina equivalents of S. cerevisiae SUP35 and SUP45 genes:
The characteristics of the mutations affecting su1 and su2 led us to suppose that they could be the equivalents of the S. cerevisiae SUP35 and SUP45 genes. These encode the cytosolic release factors, eRF3 and eRF1, respectively (FROLOVA et al. 1994 ; STANSFIELD et al. 1995 ; ZHOURAVLEVA et al. 1995 ). Proteins involved in cytosolic translation are well conserved among eukaryotic species. We thus decided to use the SUP35 and SUP45 genes as probes to screen for the P. anserina homologues.

Hybridization of P. anserina genomic DNA digested by different restriction enzymes with the SUP35 and SUP45 probes indicated that each gene has a single homologue in the P. anserina genome. Cosmid banks from the P. anserina genome were screened with the SUP35 and SUP45 probes and positive clones were obtained in both cases. One cosmid containing the homologue of SUP35 and one containing the homologue of SUP45 were used to transform strains carrying either the su1-25 or the su2-5 mutations. Complementation of the female sterility of the su1-25 mutation was observed in only the transformants obtained with the cosmid carrying the equivalent of SUP35. Similarly, complementation of female sterility of the su2-5 mutation was observed in only the transformants obtained with the cosmid carrying the equivalent of SUP45. This suggested that su1 was the homologue of SUP35 and su2 the homologue of SUP45. The restoration of female fertility of the appropriate mutant was used as criteria for subcloning both cosmids in pBC-hygro and to define the minimal DNA regions that are necessary and sufficient to ensure complementation. Two independent transgenic copies of su1 minimal complementing DNA region (T4 and T6) and two of su2 (U9 and U10) were selected for further investigations. These regions were sequenced (GenBank accession numbers are AF045014 for su1 and AF052983 for su2). Sequencing of su1 revealed a large intronless open reading frame of 715 amino acids with two in frame potential start codons. Primer extension analysis revealed that transcription starts 31 nucleotides before the most upstream initiator codon, which is in a proper context (PAIN 1996 ), strongly suggesting that this upstream codon is the inititator codon. Sequencing of su2 revealed the presence of an open reading frame interrupted by three potential introns. These were ascertained by sequencing the cDNA. The two deduced polypeptides were compared to some of the other known eRF1 or eRF3 proteins (Figure 1 and Figure 2). As seen on the figures, su1 and su2 are the bona fide homologues of SUP35 and SUP45, respectively.

su1-51 and su1-50 do not belong to the su1 gene:
As seen in Table 1, the two alleles of su1, su1-51, and su1-50, seem to display particular characteristics. Especially, unlike all the other alleles, they strongly increase longevity. To test the possibility that these two alleles belong to another closely linked gene, we have tested if a su1 transgenic copy that complements su1-25 also complements the su1-51 and su1-50 female fertility impairment. To this end, the su1-25 strain carrying the T4 transgene that was recovered in the subcloning experiment was crossed with wild type. In the progeny of this cross a su1+ strain carrying T4 was recovered. This strain was then crossed with either the su1-50 strain or the su1-51 strain. In the progeny, strains carrying the su1-51 (or su1-50) mutation and the T4 transgene were recovered. These clearly conserved the su1-51 (or su1-50) female fertility impairment. T4, while complementing su1-25, was thus not able to complement the recessive su1-50 and su1-51 mutations. This shows that the two mutations belong to a gene closely linked to but different from su1. su1-51 and su1-50 are now ascribed to the gene su15 and renamed su15-1 and su15-2, respectively.

su2 and AS2 define a unique gene:
Cloning of the su2 gene allowed us to investigate the relationship between su2 and AS2. Indeed, AS2-1 and AS2-2 might be antisuppressor alleles of su2 because they map at the same locus as su2 (PICARD-BENNOUN 1976 ). However, lack of appropriate phenotypes prevents complementation tests between su2 and AS2 alleles. AS2-1 and AS2-2 restore the female fertility of the su1-31 alleles. Based on heterokaryon tests, this effect seems to be at least partially dominant. We thus retrieved by PCR the DNA fragments containing the su2 region from the AS2-1 and AS2-2 strains. These fragments were cloned into the pBC-phleo or the pBC-hygro plasmid (SILAR 1995 ) and the resulting plasmids were transformed into the su1-31 strain. Fifty percent of the transformants in the case of AS2-1 and 3% in the case of AS2-2 are able to restore the female fertility of the su1-31 strain, showing that AS2 and su2 define a unique gene.

Sequencing of the su1 gene reveals the existence of a long N-terminal extension in eRF3 as in other organisms:
As seen in the comparison, and like the other eRF3, P. anserina eRF3 is composed of two parts, a very evolutionarily conserved carboxy terminus from position 290 to 715 (displaying around 60% similarity between the different species) and a very badly conserved N-terminal extension. Surprisingly, the C terminus of the P. anserina eRF3 lacks the last very conserved motif, "IAIGK(V/I)XKL" (X is any amino acid). The N-terminal extension is 290 amino acids long and resembles that of the two yeast proteins. As seen for these latter proteins, it is composed of two parts: (1) From amino acid 1 to 140, the sequence is very rich in glutamine/asparagine, glycine, and tyrosine. Because of the strong amino acid bias, the sequences of the three proteins are 40% identical in this region. (2) From amino acids 140 to 290, the proteins are rather divergent (they have around 20% identity) and contain high levels of charged residues. In P. anserina, the repeated sequence "AKVLSIG" is contained three times in this part of the protein. Two of these core sequences are embedded within the larger repeat, "PKAKVLSIGNPAPAK(E/D)E". A database search with these sequences revealed that they are specific to P. anserina eRF3.

Deletion of su1 is lethal:
Deletion of su1 was performed by a single step replacement of most of the coding sequence of the gene at its chromosomal location with a fragment of DNA containing a phleomycin resistance marker. This was done in a strain carrying an ectopic wild-type copy marked with a hygromycin B resistance gene (see MATERIALS AND METHODS). Crossing of the deleted strain with wild type yielded numerous two-ascospore asci, suggesting early mortality of some products of meiosis before they differentiated into ascospores. In the progeny of this cross, no homokaryotic ascospore yielding a phleo^R Hyg^S mycelium was recovered. These data show that, as in yeast (TER-AVANESYAN et al. 1993 ), deletion of the gene encoding eRF3 is lethal, but also that the presence of eRF3 is required for the morphogenesis of ascospores. From the previous cross, we recovered a heterokaryotic su1/su1⁺ strain. We have used this strain as male parent in a cross with wild type as the female partner. In the progeny, we could recover ascospores that carry the deletion showing that conidia from the heterokaryotic su1/su1⁺ strain could have the su1 genotype. su1 is likely dispensable for the formation of conidia.

Underexpression of su1 results in a suppressor phenotype:
Interestingly, some dikaryotic ascospores recovered in the crosses between the su1/su1⁺ and the su1⁺ 193 strains are green. These green ascospores have invariably the 193 su1/193 su1⁺ genotype, suggesting that roughly half the level of eRF3 promotes a suppressor phenotype. In addition, these spores have different shades of green. A possible explanation for this is the presence of different ratios of deleted versus wild-type nuclei in the ascospores (P. anserina "dikaryotic spores" possess in fact around 30 nuclei that derive from the two initial ones; D. ZICKLER, personal communication). This is confirmed by mating-type analysis. Indeed, these green spores yield strains that have a biased ratio of nuclei from one mating type versus the other (data not shown), the nucleus carrying the deletion being invariably in lower quantity. Overall, this strongly suggests that depletion of eRF3 entails a suppressor phenotype in P. anserina.

Two copies of su1 do not affect the mycelium vegetative growth and fertility but promote an antisuppressor effect:
T4 and T6, the two independent integrations of the wild-type su1 that were obtained during the subcloning of the DNA fragment encompassing su1, were used to test the effect of two copies of su1 in the same nucleus. These copies are fully functional since su1-25 strains that carry them are as fertile as wild type and have the same longevity as wild type (Table 2; Figure 3). Strains carrying either one of these ectopic copies in addition to the endogenous wild-type copy do not display any modifications of vegetative growth (including life span) and fertility (Table 2). A cross between these strains and the 193 tester strain does not yield any green spores showing that, as expected, overexpression of su1 does not promote a suppressor phenotype. To investigate whether this overexpression entails an antisuppressor phenotype, we have constructed heterokaryotic 193 su8-1 mat+/193 su8-1 mat- strains that carry su1⁺ in two copies in both nuclei. This was done by crossing a 193 su8-1 strain with the su1⁺ strains that carry either one of the T4 or T6 ectopic copies. su8-1 is a UGA suppressor tRNA_ser (DEBUCHY and BRYGOO 1985 ). These strains yield mononucleate ascospores that are less colored than the 193 su8-1 ascospores with a single copy of su1⁺, showing that two copies of su1 promote an antisuppressor phenotype.

View larger version (123K): In this window In a new window Download PPT slide   Figure 3. Macroscopic phenotype of strains carrying either a su1+ ectopic copy (T4, T6) or the "N-terminal deleted" gene (T1, T2, T7), in a su1 or su1-25 background. The arrow points toward one fruiting body (perithecium) in the control wild-type strain.

Deletion of the N terminus of eRF3 causes alterations of the sexual cycle and an unexpected suppressor phenotype:
Expression of an eRF3 protein deleted from its N terminus was achieved by introducing in a su1-25 strain a chimeric gene that fused the promoter region of su1 (including the initiator codon) to codon no. 291. Three independent transformants (T1, T2, and T7) were picked for further analysis. The su1-25 strains carrying each one of these transgenes have a wild-type vegetative mycelium, including morphology, growth speed, and longevity (Table 2). On the other hand, these strains have a slight impairment in the sexual cycle because perithecium and ascospore formations are delayed and slightly reduced (Table 2; Figure 3). This phenotype is more pronounced for T2 than T7 and for T7 than T1. For these strains, ascospore germination occurs almost as wild-type ascospores (Table 2). Crosses of these strains with the su1/su1⁺ strain then successfully introduced each one of the three transgenes into the strain carrying the deletion of su1, showing that the C terminus of the protein is sufficient to ensure viability. This yields strains that have wild-type vegetative characteristics (morphology, growth speed, and longevity; Table 2; Figure 3), showing that the N terminus of the protein is dispensable during vegetative growth. However, these strains have fertility impairment (Table 2; Figure 3), indicating that the N terminus of the protein is required for proper reproduction. This defect is more pronounced for T2 and T7 because the strains are then completely female sterile. Ascospore germination for these strains is also strongly affected (Table 2). Note that in these strains the sexual alteration is much stronger than that of the corresponding strains carrying su1-25. These data indicate that there is intragenic complementation between su1-25 and the N-terminal deletion. By crossing the su1-25 strains that carry each of the T1, T2, and T7 transgenes with the su1-26 or su1-31 strain, the deletion was recombined with the su1-26 or the su1-31 mutations to test the specificity of this phenomenon. Clearly, intragenic complementation was also observed between the N-terminal deletion and these two mutations.

Because strains deleted for su1 and carrying T2 or T7 were infertile, we could not test suppression level by using the 193 ascospore color mutation. We therefore used the auxotrophic leu1-1 mutation as a test. By crossing the su1 strains that carry each of the T4 and T6 control, or T1, T2, and T7 experimental transgenes with the leu1-1 strain we could recover strains carrying leu1-1 su1 and each of the fifth transgenes. These were assayed for growth on medium lacking leucine (Figure 4). In the control strains, we could detect a very small suppressor effect with T6 but not with T4. This suggests that T6 may not be expressed as well as T4 or the wild-type endogenous copy of su1. Suppression was detected in the three experimental strains. A clear graduation was observed, T1 presenting the smallest suppressor effect and T2 presenting the strongest. The suppressor effect was recessive (Figure 4).

View larger version (69K): In this window In a new window Download PPT slide   Figure 4. Measure of leu1-1 suppression level in strains carrying T1, T2, T7, T4, T6, and either the wild-type su1 gene or the deletion of su1. Strains of the indicated genotype were inoculated on M2 minimal medium and grown for 7 days at 27°. Diameter of the thallus obtained on this minimal medium lacking leucine is proportional to the suppression intensity (COPPIN-RAYNAL 1981 ). Unlike the leu1+ control strain, the leu1-1 control strain does not grow after 7 days on this medium. Clearly, none of the leu1-1 strains carrying the endogenous wild-type su1 gene and one transgene grow on this medium showing that no suppression occurs in these strains. On the other hand, in the su1 deletion background, T1, T2, T7, and T6 promote suppression.

Because the three experimental transgenes yielded roughly the same phenotypes but with different intensities, we tentatively tested whether the positions of integration modulated their expression. Northern blot analyses were carried on RNA extracted from growing mycelia. Data were quantified with STORM Imager, using the AS1 messenger (DEQUARD-CHEBLAT and SELLEM 1994) as an internal control. Expression of su1 was low, allowing only for rough measurements. Differences were observed between the different transgenes, T2 being more expressed (ratio of 1.5 when compared to the expression of the wild-type gene) than T1 (ratio of 1) and T7 (ratio of 0.5). Note that additional and unexpected transcripts were observed with T2 and T7 (data not shown). It is known (RAZANAMPARANY and BEGUERET 1988) that in P. anserina multiple nonconservative integration usually occurs during transformation resulting in the recovery of defective copies of the genes. The abnormal transcripts observed in T2 and T7 likely result from transcription of such aberrant integrations. The gradual fertility impairment and suppressor effect observed among the three N-terminally deleted transgenes was thus likely caused by different expression levels because of their variable integration.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We cloned the P. anserina equivalents of the S. cerevisiae SUP35 and SUP45. We showed that they correspond to the previously known su1 and su2 genes, respectively. In S. cerevisiae, the two genes encode for eRF3 and eRF1, two polypeptides that interact to yield the in vivo functional release factor of the cytosolic translation (ZHOURAVLEVA et al. 1995 ). Such an association for the corresponding polypeptides also most likely exists in P. anserina, and explains the interactions observed for mutations in the two genes (PICARD-BENNOUN 1976 ). Overall, these data strongly suggest that su1 encodes P. anserina eRF3 and su2 encodes P. anserina eRF1.

The cloning of both genes allows us to clarify the structure of the su1 and su2/AS2 loci. First, two genes are present at the su1 locus, su1 and another closely linked and unidentified gene, su15, also involved in translational accuracy control. Second, the two known antisuppressor alleles of AS2 are in fact alleles of su2.

As in yeast, deletion of su1 is lethal as expected for a subunit of the translation release factor. As in yeast, the conserved carboxy terminus of the protein is sufficient to ensure viability, suggesting that this part of the protein carries the activity. However, unlike in yeast, the amount of eRF3 is inversely correlated with the suppression level in P. anserina. In yeast, independent overexpression of either SUP35 or SUP45 does not lead to any antisuppressor phenotype, whereas simultaneous overexpression of both genes promotes such an effect (STANSFIELD et al. 1995 ). On the other hand, in humans, overexpression of eRF1 alone leads to an antisuppressor phenotype, while overexpression of eRF3 promotes transcriptional repression (LE GOFF et al. 1997 ). In P. anserina, two copies of su1 lead to an antisuppressor effect whereas in heterokaryotic ascospores containing roughly half the usual dosage of the gene, a clear suppressor effect is detected. This suggests that the modalities of the translation termination are different in yeast versus humans and Podospora. In P. anserina, the eRF3 level seems to be optimized for proper efficiency of termination and eRF3 does not seem to be present in excess.

The N terminus of the P. anserina eRF3 protein presents an unusual structure. It is composed of two parts, the most upstream portion (amino acids 1–140) having a biased amino acid composition as in yeast, and the intermediate portion (amino acids 140–297) being less charged than the corresponding yeast portion and containing long repeats that have no homology with sequences present in the database. In yeast, the upstream region is responsible for the prion phenomenon. To test the role of the N-terminal region in P. anserina, the endogenous gene was deleted and replaced with ectopic copies of either the complete gene or a gene where the two domains of the N terminus of the protein are deleted. Our data show that no physiological effect of the N terminal deletion is detected during vegetative growth, indicating that the two domains of the N terminus of the protein are dispensable during this part of the fungus lifetime. On the other hand, the sexual cycle (perithecium production, ascospore formation, and germination) is altered in the strains having the N-terminally deleted gene whereas the control strains present a normal process. This strongly suggests that at least one of the two domains of the N terminus of the protein is necessary to ensure an efficient reproductive cycle. In addition, we have detected an unexpected suppressor effect entailed by the deletion of the N-terminal part of the protein. Indeed, in yeast, a similar mutation promotes an antisuppressor phenotype (TER-AVANESYAN et al. 1993 ). This confirms that the translation termination modalities are different between yeast and P. anserina. The fact that, on the one hand, the N-terminal portion acts as a cis-activator of the C terminus of the protein and that, on the other hand, no variation in the efficiency of tRNA suppressors because of nonconventional genetic elements has been detected so far (M. PICARD, personal communication), suggests that a -like phenomenon may not exist in P. anserina. However, a final conclusion about the presence of a -like phenomenon will require more experiments.

It has previously been shown that mutations in the translational apparatus strongly affect the sexual process in P. anserina (COPPIN-RAYNAL et al. 1988 , for a review). As depicted in Table 1, mutations in su1 may drastically affect the production of perithecia as does the N-terminal deletion. In strong su1 suppressor mutations, it is most likely that general translational dysfunction is involved in sexual impairment because these mutations also display drastic vegetative growth and morphology alterations. Surprisingly, the lack of vegetative effect of the N-terminal deletion and the intragenic complementation between su1-25, su1-26, or su1-31 and the N-terminal deletion suggest that the N terminus of the protein has a particular role during the sexual process. Two contradictory observations rule out a straightforward role for the N terminus of the protein. Indeed, in the case of T1, T2, and T7, the sexual impairment is directly related to the suppression level, suggesting that both are connected. However, T6 (containing the full-length protein) displays the same suppression level as T1 (containing the deleted version). T6 allows for a wild-type efficiency of sexual reproduction whereas T1 does not, suggesting in this case that sexual impairment and suppression level are not connected. One possibility that can reconcile all the above data could be that the N terminus acts as an activator of the C terminus, mostly during the sexual process. The effect of the C terminus during reproduction would not be through suppression level control, and hence termination efficiency, but through some other function, possibly not related to translation. It was previously pointed out that the suppression level per se may not be implicated in P. anserina reproductive alteration, since fidelity mutations in different genes lead to different effects on reproduction (COPPIN-RAYNAL et al. 1988 ). Similarly, the suppression level per se does not seem to be involved in the senescence process (BELCOUR et al. 1991 ; SILAR et al. 1997 ). Alternatively, it is possible that different termination codons may respond differently to the various modifications of su1.

Finally, our data show that mutations in su1 and su2 (Table 1 and Table 2) do not promote large modifications of life span, suggesting that translational termination is not a major control point of senescence. On the other hand, most mutations of the release factors cause dramatic alteration of the sexual process. It is therefore most likely that regulation during translation termination plays a pivotal role during reproduction in P. anserina. This deserves to be explored in other organisms.

	ACKNOWLEDGMENTS

We thank Drs. M. D. TER-AVANESYAN and M. F. TUITE for the kind gift of SUP35 and SUP45 probes, Drs. C. JAMET-VIERNY for critical reading of the manuscript and M. PICARD for the kind gift of the strains. This work was supported by grants from Centre National de la Recherche Scientifique and "Aide à l'Implantation des Jeunes Equipes" from la Fondation pour la Recherche Médicale.

Manuscript received February 4, 1998; Accepted for publication May 6, 1998.

	LITERATURE CITED

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