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.

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-␤ 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. cytokine ͉ macrophage ͉ pathogenesis ͉ virulence ͉ cell wall

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.The synthesis of ribosomes is one of the major metabolic pathways of a cell. In Saccharomyces cerevisiae, ribosome assembly begins in the nucleolus after the transcription of two rRNA precursors, the 35S RNA (precursor of the 18S, 5.8S, and 25S rRNAs) and the pre-5S RNA, by RNA polymerases I and III, respectively. The synthesized pre-rRNAs are modified extensively at multiple positions specified by small nucleolar ribonucleoparticles (snoRNPs) or specific enzymes (1,22,33). During rRNA maturation, the 5Ј and 3Ј external transcribed sequences (ETS) and internal transcribed sequence 1 (ITS1) and ITS2 are removed from the 35S precursor RNA by wellordered cleavages and trimming events, which require the enzymatic activities of helicases and endo-and exonucleases (19,37).Cotranscriptional assembly of ribosomal and nonribosomal proteins in the nucleolus gives rise to a large ribonucleoprotein particle corresponding to the 90S preribosomal complexes described more than 20 years ago (35) and recently characterized biochemically (8,14). These early preribosomal complexes are further converted to smaller pre-40S (43S) and pre-60S (66S) particles, precursors of the mature small and large ribosomal subunits. The pre-40S complexes, each containing a precursor of the 18S rRNA, are exported into the cytoplasm, where they give rise to the mature 40S ribosomal particles (36). Most of the large ribosomal subunit proteins are absent from the 90S preribosomes (8,14) and associate in the nucleolus with the pre-rRNA, probably concomitantly with the formation of the pre-60S particles. During pre-60S particle maturation, 27S prerRNA intermediates are converted into 25S and 5.8S mature rRNAs by successive and well-ordered steps. Several pre-60S particles, which differ in their RNA and protein compositions,...

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 EMBO Journal (2006) IntroductionTranscription of approximately 10% of the yeast genome occurs in a cell-cycle-dependent manner (Spellman et al, 1998). For almost half of these genes, the peak of transcription takes place around START (Spellman et al, 1998;Horak et al, 2002) when cells enter a new cycle and start budding. Thus, it is not surprising that an efficient transcriptional machinery is required for the passage through START. Indeed, mutation of several factors of the transcriptional apparatus leads to defects in START and the accumulation of unbudded cells (Jahng et al, 1988;Prendergast et al, 1990;Drebot et al, 1993;Walker et al, 1997;Koch et al, 1999). Modulation of gene expression is also critical for yeast conjugation. The response to mating pheromone in Saccharomyces cerevisiae involves the transcriptional activation of roughly 200 genes (Roberts et al, 2000), which ultimately determines cell-cycle arrest in the G1 phase and the formation of mating projections.Transcription starts when specific activators bind to their cognate sites on the DNA and recruit co-activator complexes. Co-activators promote the formation of the preinitiation complex (PIC) containing general transcription factors (GTFs) and RNA polymerase II (RNAPII) at the core promoters. This event determines the initiation of transcription, which is followed by elongation and termination.Transcription through chromatin requires the action of chromatin modifying and chromatin remodeling factors. Covalent modifications of histones include phosphorylation, ubiquitylation, methylation and acetylation (Strahl and Allis, 2000;Jenuwein and Allis, 2001). Acetylation of histone N-terminal tails is generally associated with transcriptional activity and histone acetyltransferases (HAT) are recruited to promoters by transcriptional activators. Although several histone deacetylases (HDAC) contribute to transcriptional repression by interacting with specific DNA-binding repressors, recent ...