Since detection of an RNA molecule is the major criterion to define transcriptional activity, the fraction of the genome that is expressed is generally considered to parallel the complexity of the transcriptome. We show here that several supposedly silent intergenic regions in the genome of S. cerevisiae are actually transcribed by RNA polymerase II, suggesting that the expressed fraction of the genome is higher than anticipated. Surprisingly, however, RNAs originating from these regions are rapidly degraded by the combined action of the exosome and a new poly(A) polymerase activity that is defined by the Trf4 protein and one of two RNA binding proteins, Air1p or Air2p. We show that such a polyadenylation-assisted degradation mechanism is also responsible for the degradation of several Pol I and Pol III transcripts. Our data strongly support the existence of a posttranscriptional quality control mechanism limiting inappropriate expression of genetic information.

Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacological challenge. Specific oligonucleotide ligands (aptamers) can be generated for a variety of targets through the iterative evolution of a random pool of sequences (SELEX). Nuclease-resistant aptamers that recognize the human receptor tyrosine kinase RET were obtained using RET-expressing cells as targets in a modified SELEX procedure. Remarkably, one of these aptamers blocked RET-dependent intracellular signaling pathways by interfering with receptor dimerization when the latter was induced by the physiological ligand or by an activating mutation. This strategy is generally applicable to transmembrane receptors and opens the way to targeting other members of this class of proteins that are of major biomedical importance.

Cotranscriptional loading of proteins onto nascent transcripts contributes to the formation of messenger ribonucleoprotein particles (mRNPs) competent for nuclear export. The transcription machinery is believed to play a pivotal role in mRNP assembly, which is at least partially linked to the function of the THO/TREX complex and the mRNA termination/polyadenylation apparatus. Here we demonstrate a prominent role for the rate of transcription in the production of export-competent mRNPs. We show that a transcription-defective allele of the Rad3p helicase, a component of the TFIIH transcription initiation factor, suppresses several phenotypes associated with defective mRNA processing and export. Strikingly, the effects of compromised Rad3p activity can be phenocopied by a transcription elongation drug as well as by other mutations affecting transcription. Our results suggest that efficient mRNP assembly is under a kinetic control that is influenced by the rate of transcription.

In the yeast Saccharomyces cerevisiae, a common conditional phenotype associated with deletion or mutation of genes encoding mRNA export factors is the rapid accumulation of mRNAs in intranuclear foci, suggested to be near transcription sites. The nuclear RNA exosome has been implicated in retaining RNAs in these foci; on deletion of the exosome component Rrp6p, the RNA is released. To determine the exact nuclear location of retained as well as released mRNAs, we have used mRNA export mutant strains to analyze the spatial relationship between newly synthesized heat shock mRNA, the chromosomal site of transcription, and known S. cerevisiae nuclear structures such as the nucleolus and the nucleolar body. Our results show that retained SSA4 RNA localizes to an area in close proximity to the SSA4 locus. On deletion of Rrp6p and release from the genomic locus, heat shock mRNAs produced in the rat7-1 strain colocalize predominantly with nucleolar antigens. Bulk poly(A)(+) RNA, on the other hand, is localized primarily to the nuclear rim. Interestingly, the RNA binding nucleocytoplasmic shuttle protein Npl3p shows strong colocalization with bulk poly(A)(+) RNA, regardless of its nuclear location. Taken together, our data show that retention occurs close to the gene and indicate distinct nuclear fates of different mRNAs.

Several aspects of eukaryotic mRNA processing are linked to transcription. In Saccharomyces cerevisiae, overexpression of the mRNA export factor Sub2p suppresses the growth defect of hpr1 null cells, yet the protein Hpr1p and the associated THO protein complex are implicated in transcriptional elongation. Indeed, we find that a pool of heat shock HSP104 transcripts are 3'-end truncated in THO complex mutant as well as sub2 mutant backgrounds. Surprisingly, however, this defect can be suppressed by deletion of the 3'-5' exonuclease Rrp6p. This indicates that incomplete RNAs result from nuclear degradation rather than from a failure to efficiently elongate transcription. RNAs that are not degraded are retained at the transcription site in a Rrp6p-dependent manner. Interestingly, the addition of a RRP6 deletion to sub2 or to THO complex mutants shows a strong synthetic growth phenotype, suggesting that the failure to retain and/or degrade defective mRNAs is deleterious. mRNAs produced in the 3'-end processing mutants rna14-3 and rna15-2, as well as an RNA harboring a 3' end generated by a self-cleaving hammerhead ribozyme, are also retained in Rrp6p-dependent transcription site foci. Taken together, our results show that several classes of defective RNPs are subject to a quality control step that impedes release from transcription site foci and suggest that suboptimal messenger ribonucleoprotein assembly leads to RNA degradation by Rrp6p.

Several lines of evidence have implicated reactive oxygen species (ROS) in the pathogenesis of various degenerative diseases and in organismal ageing. Furthermore, it has been shown recently that the alternative pathway respiration present in plants lowers ROS mitochondrial production. An alternative oxidase (AOXp) also occurs in the filamentous fungus Podospora anserina. We show here that overexpression of this oxidase does not decrease ROS production and has no effect on longevity, mitochondrial stability or ageing in this fungus. In the same way, inactivation of the gene has no effect on these parameters. In contrast, overexpression of the alternative oxidase in the long-lived cox5::BLE mutant, deficient in cytochrome c oxidase, considerably increases ROS production of the mutant. It rescues slow growth rate and female sterility, indicating an improved energy level. This overexpression also restores senescence and mitochondrial DNA instability, demonstrating that these parameters are controlled by the energy level and not by the expression level of the alternative oxidase. We also suggest that expression of this oxidase in organisms naturally devoid of it could rescue respiratory defects resulting from cytochrome pathway dysfunctions.

Nuclear mRNA metabolism relies on the interplay between transcription, processing, and nuclear export. RNA polymerase II transcripts experience major rearrangements within the nucleus, which include alterations in the structure of the mRNA precursors as well as the addition and perhaps even removal of proteins prior to transport across the nuclear membrane. Such mRNP-remodeling steps are thought to require the activity of RNA helicases/ATPases. One such protein, the DECD box RNA-dependent ATPase Sub2p/UAP56, is involved in both early and late steps of spliceosome assembly [1-4]. Here, we report a more general function of Saccharomyces cerevisiae Sub2p in mRNA nuclear export. We observe a rapid and dramatic nuclear accumulation of poly(A)(+) RNA in strains carrying mutant alleles of sub2. Strikingly, an intronless transcript, HSP104, also accumulates in nuclei, suggesting that Sub2p function is not restricted to splicing events. The HSP104 transcripts are localized in a single nuclear focus that is suggested to be at or near their site of transcription. Intriguingly, Sub2p shows strong genetic and functional interactions with the RNA polymerase II-associated DNA/DNA:RNA helicase Rad3p as well as the nuclear RNA exosome component Rrp6p, which was independently implicated in the retention of mRNAs at transcription sites [5]. Taken together, our data suggest that Sub2p functions at an early step in the mRNA export process.

The UAP56 gene has been shown to be required for prespliceosome assembly in mammals. We report here the isolation of the Schizosaccharomyces pombe ortholog of this gene by heterologous complementation of a combined PRP40HA(3)/nam8 Delta defect in budding yeast. The Saccharomyces cerevisiae ortholog, YDL084w/SUB2, is also able to suppress this defect. We show that SUB2 is involved in splicing in vivo as well as in vitro. Sub2 defective extracts form a stalled intermediate that contains U2snRNP and can be chased into functional spliceosomes. Our experiments also suggest a role for this protein in events that precede prespliceosome formation. Data reported here as well as in the accompanying papers strongly implicate Sub2p in multiple steps of the spliceosome assembly process.

Senescence, a progressive degenerative process leading to age-related increase in mortality, is found in most eukaryotes. However, the molecular events underlying aging remain largely unknown. Understanding how longevity is regulated is a fundamental problem. Here we demonstrate that the respiratory function is a key factor that contributes to shortening lifespan of the filamentous fungus Podospora anserina . In this organism, senescence is systematically associated with mitochondrial DNA instabilities. We show that inactivation of the nuclear COX5 gene encoding subunit V of the cytochrome c oxidase complex leads to the exclusive use of the alternative respiratory pathway and to a decrease in production of reactive oxygen species. This inactivation results in a striking increase of longevity associated with stabilization of the mitochondrial chromosome. Moreover, accumulation of several senescence-specific mitochondrial DNA molecules is prevented in this nuclear mutant. These findings provide direct evidence of a causal link between mitochondrial metabolism and longevity in Podospora anserina .

ABSTRACT Podospora anserina is a filamentous fungus with a limited life span. It expresses a degenerative syndrome called senescence, which is always associated with the accumulation of circular molecules (senDNAs) containing specific regions of the mitochondrial chromosome. A mobile group II intron (α) has been thought to play a prominent role in this syndrome. Intron α is the first intron of the cytochrome c oxidase subunit I gene (COX1). Mitochondrial mutants that escape the senescence process are missing this intron, as well as the first exon of theCOX1 gene. We describe here the first mutant of P. anserina that has the α sequence precisely deleted and whose cytochrome c oxidase activity is identical to that of wild-type cells. The integration site of the intron is slightly modified, and this change prevents efficient homing of intron α. We show here that this mutant displays a senescence syndrome similar to that of the wild type and that its life span is increased about twofold. The introduction of a related group II intron into the mitochondrial genome of the mutant does not restore the wild-type life span. These data clearly demonstrate that intron α is not the specific senescence factor but rather an accelerator or amplifier of the senescence process. They emphasize the role that intron α plays in the instability of the mitochondrial chromosome and the link between this instability and longevity. Our results strongly support the idea that in Podospora, “immortality” can be acquired not by the absence of intron α but rather by the lack of active cytochromec oxidase.

Biology of the CellVolume 91, Issue 7 p. 553-553 Absence of cytochrome C oxidase leads to a striking increase of life span in podospora anserina E. Dufour, E. Dufour Centre de Génétique Moléculaire-Centre National de la Recherche Scientifique, Allée de la terrasse, 91198 Gif sur Yvette Cedex, FranceSearch for more papers by this authorJ. Boulay, J. Boulay Centre de Génétique Moléculaire-Centre National de la Recherche Scientifique, Allée de la terrasse, 91198 Gif sur Yvette Cedex, FranceSearch for more papers by this authorA. Sainsard-Chanet, A. Sainsard-Chanet Centre de Génétique Moléculaire-Centre National de la Recherche Scientifique, Allée de la terrasse, 91198 Gif sur Yvette Cedex, FranceSearch for more papers by this author E. Dufour, E. Dufour Centre de Génétique Moléculaire-Centre National de la Recherche Scientifique, Allée de la terrasse, 91198 Gif sur Yvette Cedex, FranceSearch for more papers by this authorJ. Boulay, J. Boulay Centre de Génétique Moléculaire-Centre National de la Recherche Scientifique, Allée de la terrasse, 91198 Gif sur Yvette Cedex, FranceSearch for more papers by this authorA. Sainsard-Chanet, A. Sainsard-Chanet Centre de Génétique Moléculaire-Centre National de la Recherche Scientifique, Allée de la terrasse, 91198 Gif sur Yvette Cedex, FranceSearch for more papers by this author First published: 01 February 2012 https://doi.org/10.1016/S0248-4900(99)90254-XAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume91, Issue7September 1999Pages 553-553 RelatedInformation

Tom70 and Mdm10 are mitochondrial outer membrane proteins. Tom70 is implicated in the import of proteins from the cytosol into the mitochondria in Saccharomyces cerevisiae and Neurospora crassa. Mdm10 is involved in the morphology and distribution of mitochondria in S. cerevisiae. Here we report on the characterization of the genes encoding these proteins in the filamentous fungus Podospora anserina. The two genes were previously genetically identified through a systematic search for nuclear suppressors of a degenerative process displayed by the AS1-4 mutant. The PaTom70 protein shows 80% identity with its N. crassa homolog. The PaMdm10 protein displays 35.9% identity with its S. cerevisiae homolog, and cytological analyses show that the PaMDM10-1 mutant exhibits giant mitochondria, as does the S. cerevisiae mdm10-1 mutant. Mutations in PaTOM70 and PaMDM10 result in the accumulation of specific deleted mitochondrial genomes during the senescence process of the fungus. The phenotypic properties of the single- and double-mutant strains suggest a functional relationship between the Tom70 and Mdm10 proteins. These data emphasize the role of the mitochondrial outer membrane in the stability of the mitochondrial genome in an obligate aerobe, probably through the import process.

In the filamentous fungus Podospora anserina, senescence is associated with major rearrangements of the mitochondrial DNA. The undecamer GGCGCAAGCTC has been described as a preferential site for these recombination events. We show that: (i) copies of this short sequence GGCGCAAGCTC are present in unexpectedly high numbers in the mitochondrial genome of this fungus; (ii) a short cluster of this sequence, localised in a group II intronic ORF, encodes amino acids that disrupt a protein domain that is otherwise highly conserved between various species; (iii) most of the polymorphisms observed between three related species, P.anserina, P.curvicolla and P.comata, are associated with the presence/absence of this sequence; (iv) this element lies at the boundaries of major rearrangements of the mitochondrial genomes; (v) at least two other short elements in the Podospora mitochondrial genomes display similar features. We suggest that these short elements, called MUSEs (mitochondrial ultra-short elements), could be mobile and that they contribute to evolution of the mitochondrial genome in the genus Podospora. A model for mobility involving a target DNA-primed reverse transcription step is discussed.