Genome quality control: RIP (repeat-induced point mutation) comes to Podospora.

RIP (repeat-induced point mutation) is a silencing process discovered in Neurospora crassa and so far clearly established only in this species as a currently occurring process. RIP acts premeiotically on duplicated sequences, resulting in C-G to T-A mutations, with a striking preference for CpA/TpG dinucleotides. In Podospora anserina, an RIP-like event was observed after several rounds of sexual reproduction in a strain with a 40 kb tandem duplication resulting from homologous integration of a cosmid in the mating-type region. The 9 kb sequenced show 106 C-G to T-A transitions, with 80% of the replaced cytosines located in CpA dinucleotides. This led to the alteration of at least six genes, two of which were unidentified. This RIP-like event extended to single-copy genes between the two members of the repeat. The overall data show that the silencing process is strikingly similar to a light form of RIP, unaccompanied by C-methylation. Interestingly, the N. crassa zeta-eta sequence, which acts as a potent de novo C-methylation RIP signal in this species, is weakly methylated when introduced into P. anserina. These results demonstrate that RIP, at least in light forms, can occur beyond N. crassa.

Homologous and heterologous expression of a ribosomal protein gene in Podospora anserina requires an intron.

Although the role of introns in eucaryotic nuclear genes has been much debated, it remains underinvestigated in fungi. The AS1 gene of Podospora anserina contains three introns and encodes a ribosomal protein (S12) belonging to the well-conserved bacterial S19 family. We attempted to complement the highly pleiotropic mutation AS1-4 with a cDNA encoding the homologous human (S15) protein (rig gene) under the control of the AS1 promoter. In a control experiment, the AS1+ cDNA was unable to complement fully the AS1-4 mutation. It was assumed that the AS1 cDNA was not well expressed and that the AS1 gene needed intron(s) to be efficiently expressed. Addition of the first intron of the AS1 gene to the AS1 and rig cDNAs did indeed allow complementation of all the phenotypic defects of the AS1-4 mutation. These data lead to two main conclusions. First, the human S15 ribosomal protein is functional in Podospora. Second, full expression of the Podospora AS1 gene requires at least one intron.

The S12 ribosomal protein of Podospora anserina belongs to the S19 bacterial family and controls the mitochondrial genome integrity through cytoplasmic translation.

In the filamentous fungus Podospora anserina, two nuclear genes are involved in the premature death syndrome associated with a site-specific deletion of the mitochondrial DNA: a mutant allele of the AS1 gene, encoding the cytoplasmic ribosomal protein S12, and an uncharacterized gene closely linked to the mating-type locus. We describe here the cloning and the sequencing of the wild-type and two mutant alleles of the AS1 gene. The P. anserina S12 protein belongs to the bacterial S19 ribosomal protein family and shows 72% identity with the S15 human ribosomal protein. Transformation experiments have shown that the AS1-4 mutation itself is responsible for the premature death phenotype and that it corresponds to a Gly to Asp change in the highly conserved COOH-terminal part of the protein. Use of antibodies directed against S12 did not permit detection of the mutant ribosomal protein inside the mitochondria. However, cross-reactions were observed with at least one mitochondrial ribosomal protein displaying a higher molecular weight than S12. The mitochondrial protein does not seem to be a by-product of the AS1 gene but is more likely the mitochondrial homologue of S12. These results strongly suggest that the mutant S12 protein acts indirectly to promote the mitochondrial deletion, via the cytoplasmic translation.

RPC19, the gene for a subunit common to yeast RNA polymerases A (I) and C (III).

Yeast RNA polymerases A (I) and C (III) share a subunit called AC19. The gene encoding AC19 has been isolated from yeast genomic DNA using oligonucleotide probes deduced from peptide sequences of the isolated subunit. This gene (RPC19) contains an intron-free open reading frame of 143 amino acid residues. RPC19 is a single copy gene that maps on chromosome II and is essential for cell viability. The amino acid sequence contains a sequence motif common to the Escherichia coli RNA polymerase alpha subunit, the Saccharomyces cerevisiae AC40 and B44.5 subunits, the human hRPB33 product, and the CnjC conjugation-specific gene product of Tetrahymena. The 5'-upstream region contains a sequence element, the PAC box, that has been conserved in at least 10 genes encoding subunits of RNA polymerases A and C.

At least seven ribosomal proteins are involved in the control of translational accuracy in a eukaryotic organism.

In the filamentous fungus Podospora anserina, ribosomal proteins of 60 mutants impaired in the control of translational fidelity have been submitted to electrophoretic analysis. The "four corners" system combining four different two-dimensional polyacrylamide gel electrophoretic systems has been used. An altered electrophoretic pattern has been observed for 12 mutants. In mutants su3, su12 and su11 (decreased translational fidelity), proteins S1, S7 and S8, respectively, are altered. For AS mutants (increased translational fidelity), proteins S9, S12 and S19, respectively, are altered in AS9, AS1 and AS6 mutants, and protein S29 is lacking in AS3 mutants. The data suggest that five of these genes (at least) are the structural genes for the relevant proteins (su3:S1, su12:S7, AS1:S12, AS6:S19, AS9:S9), while the AS3 gene may code for a modifying enzyme.

Different alterations of the ribosomal protein S7 lead to opposite effects on translational fidelity in the fungus Podospora anserina.

In the fungus Podospora anserina, the su12-1 mutation was previously found to decrease translational accuracy and to alter the ribosomal protein S7. The mutant protein is more basic than the wild type. Among the revertants of the two ribosomal mutations su12-1 and su12-2, 29 contained a second mutation very closely linked to su12. Biochemical analysis of these revertants by functional poly(U) tests and electrophoretical study of the ribosomes led to two conclusions. First, some revertant strains contain new mutant forms of S7. This suggests that su12 is the structural gene for the ribosomal protein S7. Second, the su12-2 revertants display antisuppressor properties in vivo and in vitro (i.e. increased translational accuracy). The electrophoretical patterns of their ribosomal proteins show new, more acidic, forms of S7. Therefore, su12 can be mutated towards either a lower or a greater translational accuracy corresponding to two opposite modifications of the global charge of the ribosomal protein S7. A more acidic form than wild type leads to increased accuracy and a more basic form to decreased accuracy.

Genetics of translational fidelity in Podospora anserina: are all the genes involved in this ribosomal function identified?

Su12-1 and su12-2 are two ribosomal suppressor mutations previously described in the fungus Podospora anserina. Revertants were isolated on the criteria of either improved growth at 27 degrees C (for su12-1) or suppression of the paromomycin hypersensitivity (for su12-2). Among 45 mutations lying outside the su12 locus, only one was found which defines a new antisuppressor locus, AS9. About 3/4 of these mutations are antisuppressor mutations localized in the previously identified AS6 and AS7 genes. While the AS6 mutations harbour diverse phenotypes, all the mutations lying in the AS7 gene lead to the same phenotypic alterations. In addition, two new su3 mutations were obtained and shown to display an antisuppressor effect on su12-1.

Identification of the structural gene for the S9 ribosomal protein in the fungus Podospora anserina: a new protein involved in the control of translational accuracy.

AS9-1 was isolated as a mutation restoring growth in a strain carrying the ribosomal mutation su12-1. The AS9-1 mutation confers a weak antisuppressor effect and a low level of resistance to paromomycin. Two-dimensional polyacrylamide gel electrophoresis patterns of the ribosomal proteins from AS9-1 strains show an altered S9 protein which is more basic than the wild-type form. The presence of the two forms of the protein (wild-type and mutant) in heterocaryotic strains strongly suggests that AS9 is the structural gene for the ribosomal protein S9.