[Show abstract][Hide abstract] ABSTRACT: Nonsense-mediated mRNA decay (NMD) is a translation-dependent RNA quality-control pathway targeting transcripts such as messenger RNAs harboring premature stop-codons or short upstream open reading frame (uORFs). Our transcription start sites (TSSs) analysis of Saccharomyces cerevisiae cells deficient for RNA degradation pathways revealed that about half of the pervasive transcripts are degraded by NMD, which provides a fail-safe mechanism to remove spurious transcripts that escaped degradation in the nucleus. Moreover, we found that the low specificity of RNA polymerase II TSSs selection generates, for 47% of the expressed genes, NMD-sensitive transcript isoforms carrying uORFs or starting downstream of the ATG START codon. Despite the low abundance of this last category of isoforms, their presence seems to constrain genomic sequences, as suggested by the significant bias against in-frame ATGs specifically found at the beginning of the corresponding genes and reflected by a depletion of methionines in the N-terminus of the encoded proteins.
[Show abstract][Hide abstract] ABSTRACT: Nonsense-mediated mRNA decay (NMD) destabilizes eukaryotic transcripts with long 3' UTRs. To investigate whether other transcript features affect NMD, we generated yeast strains expressing chromosomal-derived mRNAs with 979 different promoter and open reading frame (ORF) regions and with the same long, destabilizing 3' UTR. We developed a barcode-based DNA microarray strategy to compare the levels of each reporter mRNA in strains with or without active NMD. The size of the coding region had a significant negative effect on NMD efficiency. This effect was not specific to the tested 3' UTR because two other different NMD reporters became less sensitive to NMD when ORF length was increased. Inefficient NMD was not due to a lack of association of Upf1 to long ORF transcripts. In conclusion, in addition to a long 3' UTR, short translation length is an important feature of NMD substrates in yeast.
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic genomes are pervasively transcribed. However, it is unclear how many newly found RNAs have functions and how many are byproducts of functional, or spurious, transcription events. Cells control the accumulation of many opportunistic transcripts by limiting their synthesis and by provoking their early transcription termination and decay. In this review, we use S. cerevisiae and mammalian cells as models to discuss the circumstances by which pervasive transcripts are produced and turned over. This ultimately relates to the likelihood, and potential mechanism, of molecular function.
[Show abstract][Hide abstract] ABSTRACT: Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24,
suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal
proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing
that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed
that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing
sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7Lb within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different
positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure
of ITS2 during 60S ribosomal subunit maturation.
Full-text · Article · Aug 2013 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Ribosome stalling on eukaryotic mRNAs triggers cotranslational RNA and protein degradation through conserved mechanisms. For example, mRNAs lacking a stop codon are degraded by the exosome in association with its cofactor, the SKI complex, whereas the corresponding aberrant nascent polypeptides are ubiquitinated by the E3 ligases Ltn1 and Not4 and become proteasome substrates. How translation arrest is linked with polypeptide degradation is still unclear. Genetic screens with SKI and LTN1 mutants allowed us to identify translation-associated element 2 (Tae2) and ribosome quality control 1 (Rqc1), two factors that we found associated, together with Ltn1 and the AAA-ATPase Cdc48, to 60S ribosomal subunits. Translation-associated element 2 (Tae2), Rqc1, and Cdc48 were all required for degradation of polypeptides synthesized from Non-Stop mRNAs (Non-Stop protein decay; NSPD). Both Ltn1 and Rqc1 were essential for the recruitment of Cdc48 to 60S particles. Polysome gradient analyses of mutant strains revealed unique intermediates of this pathway, showing that the polyubiquitination of Non-Stop peptides is a progressive process. We propose that ubiquitination of the nascent peptide starts on the 80S and continues on the 60S, on which Cdc48 is recruited to escort the substrate for proteasomal degradation.
Full-text · Article · Mar 2013 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: The exosome is a complex involved in the maturation of rRNA and sn-snoRNA, in the degradation of short-lived noncoding RNAs, and in the quality control of RNAs produced in mutants. It contains two catalytic subunits, Rrp6p and Dis3p, whose specific functions are not fully understood. We analyzed the transcriptome of combinations of Rrp6p and Dis3p catalytic mutants by high-resolution tiling arrays. We show that Dis3p and Rrp6p have both overlapping and specific roles in degrading distinct classes of substrates. We found that transcripts derived from more than half of intron-containing genes are degraded before splicing. Surprisingly, we also show that the exosome degrades large amounts of tRNA precursors despite the absence of processing defects. These results underscore the notion that large amounts of RNAs produced in wild-type cells are discarded before entering functional pathways and suggest that kinetic competition with degradation proofreads the efficiency and accuracy of processing.
[Show abstract][Hide abstract] ABSTRACT: Mots clefs : Biologie, Analyse de cribles et de données, Hiérarchisation, Clusterisation. Le protéasome est une protéase multimérique composée de différents sous-complexes obtenus par l'assemblage ordonné de dizaines de polypeptides. Il est présent chez toutes les cellules eucaryotes où il constitue l'unité catalytique du système Ubiquitine-protéasome (UPS). Son activité de dégradation des protéines, à la fois cytoplasmique et nucléaire est essentielle à de nombreux processus cellulaires . Le protéasome est une machinerie plastique et dynamique qui est elle-même soumise à régulation . Des dysfonctionnements de l'UPS participent à la pathogenèse de maladies telles que les cancers ou les maladies neurodégénératives. Il semble donc important d'identifier i) les processus cellulaires dans lesquels le protéasome est impliqué, ii) les facteurs influençant son activité. Dans ce but, nous développons un projet pour identifier et caractériser les partenaires physiques et fonctionnels du protéasome par une approche multi-technique chez la levure S. cerevisiae. Nous déterminons ensuite s'ils sont fonctionnellement conservés chez les Mammifères. Ce projet peut être divisé en trois étapes : 1/ L'identification de partenaires La première étape consiste à identifier des partenaires physiques et fonctionnels du protéasome par la réalisation de cribles à grande échelle utilisant le modèle cellulaire Saccharomyces cerevisiae. 2/L'exploitation des données grâce au logiciel R L'utilisation du logiciel R permet d'exploiter les résultats des cribles et de croiser ces données avec d'autres données d'interactions issues de résultats du laboratoire ou de ressources bibliographiques. Les cribles utilisés pour identifier les partenaires fonctionnels du protéasome reposent sur la technologie des puces à ADN. La quantité des données quantitatives qui en sont issues doit être gérée et exploitée pour contrôler la reproductibilité des cribles et distinguer les répondants du bruit de fond. Les résultats de ses cribles peuvent ensuite être hiérarchisés pour essayer d'identifier des groupes répondants de façon similaire ou opposé. Le deuxième aspect de l'utilisation du logiciel R dans ce projet est le regroupement de données expérimentales d'origines variées mais ayant pour point commun l'étude des partenaires du protéasome. L'intérêt de cette approche est double : i) chaque technique expérimentale présente des biais et bruits de fond différents, comparer des résultats de techniques très différentes permet de s'affranchir du bruit de fond de chacune et d'être plus sensible ; ii) les techniques à grande échelle, qui sont de plus en plus utilisées, génèrent une très grande quantité de données qu'il est impossible de mémoriser et comparer aux données existantes antérieures sans outil informatique, compiler ces données grâce au logiciel R permet de les pérenniser et de pouvoir donner du poids à des données qui ont peu de poids isolées mais qui deviennent significatives quand elles sont retrouvées dans d'autres jeux de données.
[Show abstract][Hide abstract] ABSTRACT: Hydrogen selenide is a recurrent metabolite of selenium compounds. However, few experiments studied the direct link between this toxic agent and cell death. To address this question, we first screened a systematic collection of Saccharomyces cerevisiae haploid knockout strains for sensitivity to sodium selenide, a donor for hydrogen selenide (H(2)Se/HSe(-/)Se(2-)). Among the genes whose deletion caused hypersensitivity, homologous recombination and DNA damage checkpoint genes were over-represented, suggesting that DNA double-strand breaks are a dominant cause of hydrogen selenide toxicity. Consistent with this hypothesis, treatment of S. cerevisiae cells with sodium selenide triggered G2/M checkpoint activation and induced in vivo chromosome fragmentation. In vitro, sodium selenide directly induced DNA phosphodiester-bond breaks via an O(2)-dependent reaction. The reaction was inhibited by mannitol, a hydroxyl radical quencher, but not by superoxide dismutase or catalase, strongly suggesting the involvement of hydroxyl radicals and ruling out participations of superoxide anions or hydrogen peroxide. The (•)OH signature could indeed be detected by electron spin resonance upon exposure of a solution of sodium selenide to O(2). Finally we showed that, in vivo, toxicity strictly depended on the presence of O(2). Therefore, by combining genome-wide and biochemical approaches, we demonstrated that, in yeast cells, hydrogen selenide induces toxic DNA breaks through an O(2)-dependent radical-based mechanism.
[Show abstract][Hide abstract] ABSTRACT: Correlation between the relative fitness defects associated with sodium selenide and Cpt treatments. The data of the present study were compared with the fitness scores associated with Cpt treatment . The comparison involves 4386 genes. The gray area corresponds to genes whose invalidation leads to fitness parameters smaller than −0.5 in the cases of the two toxic compounds. This comparison indicates a good correlation between the behaviors of the mutants in response to the two toxic agents (non-parametric Kendall test, p = 3.2·10−14). 1. Hillenmeyer ME, Fung E, Wildenhain J, Pierce SE, Hoon S, et al. (2008) The chemical genomic portrait of yeast: uncovering a phenotype for all genes. Science 320∶362–365.
[Show abstract][Hide abstract] ABSTRACT: Strategy for quantitative analysis of a cell population using a two-step amplification of DNA barcodes. Barcode regions are in gray. In the first PCR, one oligonucleotide hybridizes to a sequence beyond the barcode region (U1 for the upstream barcode, D1 for the downstream one) and the other one hybridizes inside the kanMX cassette (KU and KD, respectively). The second amplification is performed in the presence of fluorescent primers U2/D2 annealing inside the first amplicons, and in the presence of unlabeled U1/D1 primers. The PCR products (and thus the corresponding barcodes) can then quantified by microarray hybridization.
[Show abstract][Hide abstract] ABSTRACT: Fitness defect analysis. (A) Distribution of the relative fitness defect values (log2(s)) obtained with 4520 mutants of the systematic deletion collection. The observed distribution of fitness defect values (bins of size 0.01) was fitted to a Cauchy-Lorentz function by non-linear least squares regression. Part of the histogram and the fitted values are shown in the inset to illustrate the asymmetry of the results distribution, with an imbalance towards negative values (corresponding to strains that grew poorly in the presence of Na2Se). (B) Comparison of observed fitness defects obtained on pairs of strains that are deleted at the same locus. Data include 294 pairs of mutants for which there is an overlap of at least one nucleotide between the two corresponding deleted ORFs. An example is given at the top of the panel, where ORF YBR098W (MMS4) fully overlaps ORF YBR099C. The relative fitness defects observed for such pairs are represented as a scatter plot, arbitrarily assigning the mutated alleles to the X or the Y axis. The graph indicates high correlation between fitness values for each pair of mutants (non-parametric Kendall test, p<2.2·10−16).
[Show abstract][Hide abstract] ABSTRACT: RNA Pol II transcription termination can occur by at least two alternative pathways. Cleavage and polyadenylation by the CPF/CF complex precedes mRNA transcription termination, while the Nrd1 complex is involved in transcription termination of non-coding RNAs such as sno/snRNAs or cryptic unstable transcripts. Here we show that transcription of RPL9B, one of the two genes coding for the ribosomal protein Rpl9p, terminates by either of these two pathways. The balance between these two pathways is modulated in response to the RPL9 gene copy number, resulting in the autoregulation of RPL9B gene expression. This autoregulation mechanism requires a conserved potential stem-loop structure very close to the polyadenylation sites. We propose a model in which Rpl9p, when in excess, binds this conserved 3'-UTR structure, negatively interfering with cleavage and polyadenylation to the benefit of the Nrd1-dependent termination pathway, which, being coupled to degradation by the nuclear exosome, results in downregulation of RPL9B gene expression.
[Show abstract][Hide abstract] ABSTRACT: The EKC/KEOPS complex is universally conserved in Archaea and Eukarya and has been implicated in several cellular processes, including transcription, telomere homeostasis and genomic instability. However, the molecular function of the complex has remained elusive so far. We analyzed the transcriptome of EKC/KEOPS mutants and observed a specific profile that is highly enriched in targets of the Gcn4p transcriptional activator. GCN4 expression was found to be activated at the translational level in mutants via the defective recognition of the inhibitory upstream ORFs (uORFs) present in its leader. We show that EKC/KEOPS mutants are defective for the N6-threonylcarbamoyl adenosine modification at position 37 (t(6)A(37)) of tRNAs decoding ANN codons, which affects initiation at the inhibitory uORFs and provokes Gcn4 de-repression. Structural modeling reveals similarities between Kae1 and bacterial enzymes involved in carbamoylation reactions analogous to t(6)A(37) formation, supporting a direct role for the EKC in tRNA modification. These findings are further supported by strong genetic interactions of EKC mutants with a translation initiation factor and with threonine biosynthesis genes. Overall, our data provide a novel twist to understanding the primary function of the EKC/KEOPS and its impact on several essential cellular functions like transcription and telomere homeostasis.
Full-text · Article · Apr 2011 · Nucleic Acids Research
[Show abstract][Hide abstract] ABSTRACT: Cryptic unstable transcripts (CUTs) have been recently described as a major class of non-coding RNAs. These transcripts are, however, extremely unstable in normal cells and their analyzes pose specific technical problems. In this chapter, after a brief introduction discussing general aspects associated with the analysis of non-coding RNAs, we provide details of methods to enrich, map, and quantify this unconventional class of transcripts.
No preview · Article · Jan 2011 · Methods in molecular biology (Clifton, N.J.)
[Show abstract][Hide abstract] ABSTRACT: In eukaryotes, ribosome biogenesis is a highly conserved process that starts in the nucleus and ends in the cytoplasm. In actively growing yeast cells, it is estimated that each nuclear pore complex (NPC) contributes to the export of about 25 pre-ribosomal particles per minute. Such an extremely active process requires several redundant export receptors for the pre-60S particles. Here, we report the identification of a novel pre-60S factor, Ecm1, which partially acts like Arx1 and becomes essential when the NPC function is affected. Ecm1 depletion, combined with the deletion of NPC components led to pre-60S retention in the nucleus. Functional links that we identified between Ecm1, 60S biogenesis, pre-60S export, and the NPC were correlated with physical interactions of Ecm1 with pre-60S particles and nucleoporins. These results support that Ecm1 is an additional factor involved in pre-60S export. While Ecm1 and Arx1 have redundant functions, overproduction of either one could not complement the absence of the other, whereas overproduction of Mex67 was able to partially restore the growth defect resulting from the absence of Ecm1 or Arx1. These data highlight the involvement of many factors acting together to export pre-60S particles.
[Show abstract][Hide abstract] ABSTRACT: Over the past few years, techniques have been developed that have allowed the study of transcriptomes without bias from previous genome annotations, which has led to the discovery of a plethora of unexpected RNAs that have no obvious coding capacities. There are many different kinds of products that are generated by this pervasive transcription; this Review focuses on small non-coding RNAs (ncRNAs) that have been found to be associated with promoters in eukaryotes from animals to yeast. After comparing the different classes of such ncRNAs described in various studies, the Review discusses how the models proposed for their origins and their possible functions challenge previous views of the basic transcription process and its regulation.
Preview · Article · Dec 2009 · Nature Reviews Genetics