Jocelyne Boulay

Cryptic Pol II Transcripts Are Degraded by a Nuclear Quality Control Pathway Involving a New Poly(A) Polymerase

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.

Interactions between mRNA Export Commitment, 3′-End Quality Control, and Nuclear Degradation

ABSTRACT 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.

Neutralizing aptamers from whole-cell SELEX inhibit the RET receptor tyrosine kinase.

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.

The DECD box putative ATPase Sub2p is an early mRNA export factor

Abstract 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.

Overexpression of the alternative oxidase restores senescence and fertility in a long-lived respiration-deficient mutant of Podospora anserina

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.

Mutations in genes encoding the mitochondrial outer membrane proteins Tom70 and Mdm10 of Podospora anserina modify the spectrum of mitochondrial DNA rearrangements associated with cellular death.

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.

Mitochondrial Group II Introns, Cytochrome c Oxidase, and Senescence 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.

In vivo SELEX reveals novel sequence and structural determinants of Nrd1-Nab3-Sen1-dependent transcription termination.

The Nrd1‐Nab3‐Sen1 (NNS) complex pathway is responsible for transcription termination of cryptic unstable transcripts and sn/snoRNAs. The NNS complex recognizes short motifs on the nascent RNA, but the presence of these sequences alone is not sufficient to define a functional terminator. We generated a homogeneous set of several hundreds of artificial, NNS‐dependent terminators with an in vivo selection approach. Analysis of these terminators revealed novel and extended sequence determinants for transcription termination and NNS complex binding as well as supermotifs that are critical for termination. Biochemical and structural data revealed that affinity and specificity of RNA recognition by Nab3p relies on induced fit recognition implicating an α‐helical extension of the RNA recognition motif. Interestingly, the same motifs can be recognized by the NNS or the mRNA termination complex depending on their position relative to the start of transcription, suggesting that they function as general transcriptional insulators to prevent interference between the non‐coding and the coding yeast transcriptomes.

Intramolecular cross-overs generate deleted mitochondrial DNA molecules in Podospora anserina

Abstract The unavoidable senescence process that limits the vegetative growth of Podospora anserina is always associated with an accumulation of various classes of circular, tandemly arranged, defective mitochondrial DNA molecules (senDNAs). The monomers of the senDNAs belonging to the so-called β class share a common core, but differ in both their length and termini. To understand the mechanism leading to their formation, we have determined the junction sequence of 36 senDNA β monomers present in various senescent cultures. In most cases, we observe that: (1) short direct repeats precisely bound the senDNA β termini and (2) one copy of the repeats is retained in the senDNA sequence. Moreover, PCR analysis of the mitochondrial DNA of some of the senescent cultures, has allowed us to detect another genome which is exactly lacking the sequence of the senDNA β found in the culture. These results demonstrate that an intramolecular unequal cross-over occurring between short direct repeats can generate deleted mtDNA molecules in P. anserina. In addition, the polymorphism displayed by one pair of repeats allows us to establish that this cross-over may be associated with a short conversion tract spanning a few (about 15) nucleotides.

Contribution of various classes of defective mitochondrial DNA molecules to senescence in Podospora anserina.

Abstract The unavoidable arrest of vegetative growth in Podospora anserina (senescence process) is always correlated with rearrangements of the mitochondrial chromosome, mainly consisting in the amplification of particular regions as tandemly repeated circular molecules (senDNAs). One sequence systematically amplified in senescent cultures corresponds precisely to the first intron (intron α) of the cox1 gene; nevertheless, other regions (called β and γ) are also frequently amplified. The experiments presented in this paper show that cellular death is in some cases associated with the sole presence of large amounts of senDNA β. In addition, we provide evidence that senDNA β and senDNA α accumulate by different mechanisms, as previously proposed. This suggests that β senDNAs have a lethal effect on the mycelium on their own and most likely have replicative properties independent of the presence of sequence α. These data do not fit well with the current opinion that gives an essential role to intron α in the senescence of P. anserina.