Jean-Claude Rousselle

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.

A critical role for peptidoglycan N-deacetylation in Listeria evasion from the host innate immune system

Listeria monocytogenes is a human intracellular pathogen that is able to survive in the gastrointestinal environment and replicate in macrophages, thus bypassing the early innate immune defenses. Peptidoglycan (PG) is an essential component of the bacterial cell wall readily exposed to the host and, thus, an important target for the innate immune system. Characterization of the PG from L. monocytogenes demonstrated deacetylation of N-acetylglucosamine residues. We identified a PG N-deacetylase gene, pgdA, in L. monocytogenes genome sequence. Inactivation of pgdA revealed the key role of this PG modification in bacterial virulence because the mutant was extremely sensitive to the bacteriolytic activity of lysozyme, and growth was severely impaired after oral and i.v. inoculations. Within macrophage vacuoles, the mutant was rapidly destroyed and induced a massive IFN-β response in a TLR2 and Nod1-dependent manner. Together, these results reveal that PG N-deacetylation is a highly efficient mechanism used by Listeria to evade innate host defenses. The presence of deacetylase genes in other pathogenic bacteria indicates that PG N-deacetylation could be a general mechanism used by bacteria to evade the host innate immune system.

Sequential Protein Association with Nascent 60S Ribosomal Particles

ABSTRACT Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood. We show here that the association of preribosomal factors with pre-60S complexes depends on the presence of earlier factors, a phenomenon essential for ribosome biogenesis. The analysis of the composition of purified preribosomal complexes blocked in maturation at specific steps allowed us to propose a model of sequential protein association with, and dissociation from, early pre-60S complexes for several preribosomal factors such as Mak11, Ssf1, Rlp24, Nog1, and Nog2. The presence of either Ssf1 or Nog2 in complexes that contain the 27SB pre-rRNA defines novel, distinct pre-60S particles that contain the same pre-rRNA intermediates and that differ only by the presence or absence of specific proteins. Physical and functional interactions between Rlp24 and Nog1 revealed that the assembly steps are, at least in part, mediated by direct protein-protein interactions.

AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX.

Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase‐independent mode of cell death mediated by apoptosis‐inducing factor (AIF). After poly(ADP‐ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C‐terminal proline‐rich binding domain (PBD, residues 543–559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA‐degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C‐terminal PBD abolishes AIF/H2AX interaction and AIF‐mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA‐deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase‐independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.

Yeast homolog of a cancer‐testis antigen defines a new transcription complex

We have isolated a new yeast gene (PCC1) that codes for a factor homologous to human cancer‐testis antigens. We provide evidence that Pcc1p is a new transcription factor and that its mutation affects expression of several genes, some of which are involved in cell cycle progression and polarized growth. Mutation of Pcc1p also affects the expression of GAL genes by impairing the recruitment of the SAGA and Mediator co‐activators. We characterize a new complex that contains Pcc1p, a kinase, Bud32p, a putative endopeptidase, Kae1p and two additional proteins encoded by ORFs YJL184w and YMLO36w. Genetic and physical interactions among these proteins strongly suggest that this complex is a functional unit. Chromatin immunoprecipitation experiments and multiple genetic interactions of pcc1 mutants with mutants of the transcription apparatus and chromatin modifying enzymes underscore the direct role of the complex in transcription. Functional complementation experiments indicate that the transcriptional function of this set of genes is conserved throughout evolution.

The ribosomal protein Rps15p is required for nuclear exit of the 40S subunit precursors in yeast

We have conducted a genetic screen in order to identify ribosomal proteins of Saccharomyces cerevisiae involved in nuclear export of the small subunit precursors. This has led us to distinguish Rps15p as a protein dispensable for maturation of the pre‐40S particles, but whose assembly into the pre‐ribosomes is a prerequisite to their nuclear exit. Upon depletion of Rps15p, 20S pre‐rRNA is released from the nucleolus and retained in the nucleus, without alteration of the pre‐rRNA early cleavages. In contrast, Rps18p, which contacts Rps15p in the small subunit, is required upstream for pre‐rRNA processing at site A2. Most pre‐40S specific factors are correctly associated with the intermediate particles accumulating in the nucleus upon Rps15p depletion, except the late‐binding proteins Tsr1p and Rio2p. Here we show that these two proteins are dispensable for nuclear exit; instead, they participate in 20S pre‐rRNA processing in the cytoplasm. We conclude that, during the final maturation steps in the nucleus, incorporation of the ribosomal protein Rps15p is specifically required to render the pre‐40S particles competent for translocation to the cytoplasm.

In Vivo Interactome of Helicobacter pylori Urease Revealed by Tandem Affinity Purification

In the human gastric bacterium Helicobacter pylori, two metalloenzymes, hydrogenase and urease, are essential for in vivo colonization, the latter being a major virulence factor. The UreA and UreB structural subunits of urease and UreG, one of the accessory proteins for Ni2+ incorporation into apourease, were taken as baits for tandem affinity purification. The method allows the purification of protein complexes under native conditions and physiological expression levels of the bait protein. Furthermore the tandem affinity purification technology was combined with in vivo cross-link to capture transient interactions. The results revealed different populations of urease complexes: (i) urease captured during activation by Ni2+ ions comprising all the accessory proteins and (ii) urease in association with metabolic proteins involved e.g. in ammonium incorporation and the cytoskeleton. Using UreG as a bait protein, we copurified HypB, the accessory protein for Ni2+ incorporation into hydrogenase, that is reported to play a role in urease activation. The interactome of HypB partially overlapped with that of urease and revealed interactions with SlyD, which is known to be involved in hydrogenase maturation as well as with proteins implicated in the formation of [Fe-S] clusters present in the small subunit of hydrogenase. In conclusion, this study provides new insight into coupling of ammonium production and assimilation in the gastric pathogen and the intimate link between urease and hydrogenase maturation.

Linking functionally related genes by sensitive and quantitative characterization of genetic interaction profiles

Describing at a genomic scale how mutations in different genes influence one another is essential to the understanding of how genotype correlates with phenotype and remains a major challenge in biology. Previous studies pointed out the need for accurate measurements of not only synthetic but also buffering interactions in the characterization of genetic networks and functional modules. We developed a sensitive and efficient method that allows such measurements at a genomic scale in yeast. In a pilot experiment (41 genome-wide screens), we quantified the fitness of 140,000 double deletion strains relative to the corresponding single mutants and identified many genetic interactions. In addition to synthetic growth defects (validated experimentally with factors newly identified as genetically interfering with mRNA degradation), most of the identified genetic interactions measured weak epistatic effects. These weak effects, rarely meaningful when considered individually, were crucial to defining specific signatures for many gene deletions and had a major contribution in defining clusters of functionally related genes.

Phosphoproteomic analysis of Leishmania donovani pro- and amastigote stages.

Following transmission to the vertebrate host, the protozoan parasite Leishmania donovani differentiates into the pathogenic amastigote stage that is adapted for intracellular survival. This developmental transition is induced by environmental factors including elevated temperature and acidic pH and is likely transduced by signaling cascades involving protein kinases and their downstream phosphoprotein substrates. These signaling networks are highly adapted to the specific nutritional and physiological requirements of the organism and thus studying Leishmania phosphorylation may allow important insight into the parasite‐specific biology. We used a gel‐based approach to investigate qualitative and quantitative changes of the phosphoproteome of the major L. donovani life cycle stages. Phosphoproteins were purified by immobilized metal affinity chromatography (IMAC), separated by IEF and SDS‐PAGE using pH 4–7 IPG immobiline strips, revealed by fluorescent multiplex staining, and identified by MALDI‐MS and MS/MS. Our analysis allowed us to establish a first repertoire of the Leishmania phosphoproteome and to identify phosphoproteins implicated in stress‐ and heat shock response, RNA/protein turnover, metabolism, and signaling.

Proteolytic Regulation of the Urokinase Receptor/CD87 on Monocytic Cells by Neutrophil Elastase and Cathepsin G

The urokinase receptor (CD87) participates to the pericellular proteolytic potential of migrating cells and to the recruitment of leukocytes during inflammation. It consists of three structurally homologous domains, with the C-terminal domain D3 attached to cell membranes through a GPI anchor. CD87 is susceptible to an endoproteolytic processing removing the N-terminal domain D1 and generating truncated D2D3 membrane species, thus modulating CD87-associated functions. Full-length or truncated CD87 can be also released from cells via juxtamembrane cleavage by phospholipases and/or by yet unidentified proteinases. Using a recombinant CD87 and the CD87-positive monocytic U937 cell line and isolated blood monocytes, we show by protein immunoblotting and flow immunocytometry that the human neutrophil serine-proteinases elastase and cathepsin G cleave CD87 within the D1-D2 linker sequence, while in addition cathepsin G is highly efficient in cleaving the C terminus of D3. The combination of cathepsin G and elastase provided by degranulated neutrophils results in enzymatic cooperation leading to the release from monocytic cells of a truncated D2D3 species resembling that previously described in pathological body fluids. Using mass spectrometry analysis, the proteolytic fragmentation of synthetic peptides mapping the D1-D2 linker and D3 C-terminal domains identifies potential cleavage sites for each enzyme and suggests the existence of a mechanism regulating the CD87(D1-D2)-associated chemotactic activity. Finally, isolated or combined elastase and cathepsin G drastically reduce the capacity of cells to bind urokinase. Secretable leukocyte serine-proteinases are thus endowed with high potential for the regulation of CD87 expression and function on inflammatory cells.