Publications (19)83.33 Total impact
- [Show abstract] [Hide abstract] ABSTRACT: Ribosome profiling is an emerging approach using deep sequencing of the mRNA part protected by the ribosome to study protein synthesis at the genome scale. This approach provides new insights into gene regulation at the translational level. In this review we describe the protocol to prepare polysomes and extract ribosome protected fragments before to deep sequence them.
- [Show abstract] [Hide abstract] ABSTRACT: The universal tRNA modification t6A is found at position 37 of nearly all tRNAs decoding ANN codons. The absence of t6A37 leads to severe growth defects in baker’s yeast, phenotypes similar to those caused by defects in mcm5s2U34 synthesis. Mutants in mcm5s2U34 can be suppressed by overexpression of tRNALysUUU, but we show t6A phenotypes could not be suppressed by expressing any individual ANN decoding tRNA, and t6A and mcm5s2U are not determinants for each other’s formation. Our results suggest that t6A deficiency, like mcm5s2U deficiency, leads to protein folding defects, and show that the absence of t6A led to stress sensitivities (heat, ethanol, salt) and sensitivity to TOR pathway inhibitors. Additionally, L-homoserine suppressed the slow growth phenotype seen in t6A-deficient strains, and proteins aggregates and Advanced Glycation End-products (AGEs) were increased in the mutants. The global consequences on translation caused by t6A absence were examined by ribosome profiling. Interestingly, the absence of t6A did not lead to global translation defects, but did increase translation initiation at upstream non-AUG codons and increased frame-shifting in specific genes. Analysis of codon occupancy rates suggests that one of the major roles of t6A is to homogenize the process of elongation by slowing the elongation rate at codons decoded by high abundance tRNAs and I34:C3 pairs while increasing the elongation rate of rare tRNAs and G34:U3 pairs. This work reveals that the consequences of t6A absence are complex and multilayered and has set the stage to elucidate the molecular basis of the observed phenotypes.
- [Show abstract] [Hide abstract] ABSTRACT: Ribosome profiling provides genome-wide information about translational regulation. However, there is currently no standard tool for the qualitative analysis of Ribo-seq data. We present here RiboTools, a Galaxy toolbox for the analysis of ribosome profiling (Ribo-seq) data. It can be used to detect translational ambiguities, stop codon readthrough events and codon occupancy. It provides a large number of plots for the visualisation of these events. RiboTools is available from https://testtoolshed.g2.bx.psu.edu/view/rlegendre/ribo_tools as part of the Galaxy Project, under the GPLv3 licence. It is written in python2.7 and uses standard python libraries, such as matplotlib and numpy. firstname.lastname@example.org SUPPLEMENTARY INFORMATION: Supplementary materials are available from Bioinformatics online. © The Author (2015). Published by Oxford University Press. All rights reserved. For Permissions, please email: email@example.com.
- [Show abstract] [Hide abstract] ABSTRACT: Prions are infectious proteins that can adopt a structural conformation that is then propagated among other molecules of the same protein. [PSI(+)] is an aggregated conformation of the translational release factor eRF3. [PSI(+)] modifies cellular fitness, inducing various phenotypes depending on genetic background. However, the genes displaying [PSI(+)]-controlled expression remain unknown. We used ribosome profiling in isogenic [PSI(+)] and [psi(-)] strains to identify the changes induced by [PSI(+)]. We found 100 genes with stop codon readthrough events and showed that many stress-response genes were repressed in the presence of [PSI(+)]. Surprisingly, [PSI(+)] was also found to affect reading frame selection independently of its effect on translation termination efficiency. These results indicate that [PSI(+)] has a broader impact than initially anticipated, providing explanations for the phenotypic differences between [psi(-)] and [PSI(+)] strains.
- [Show abstract] [Hide abstract] ABSTRACT: The [PSI (+) ] determinant in Saccharomyces cerevisiae is the prion protein corresponding to the eRF3 translation termination factor. Numerous infectious proteins have been described in yeast, in comparison of the unique PrP protein in higher eukaryotes. The presence of the PrP prion is associated with mammalian diseases. Whether fungal prions are beneficial or deleterious are still under discussions. The review focuses on [PSI (+) ] -induced phenotypes and the resulting physiological consequences to shed light on the cellular changes occurring in a [PSI (+) ] cell and its possible role in nature. To date, only two genes directly regulated at the translational level by [PSI (+) ] have been identified. Yet, through all the published works, obtaining a consensus for the described [PSI (+) ] phenotypes appeared a tricky task. They are highly dependent on the prion variant and the genetic background of the strain. The [PSI (+) ] prion might generate diverse modifications not only at the translational, but also at the transcriptional levels, and the phenotypic heterogeneity is the result of these complex combinations of the genotypic 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.
- [Show abstract] [Hide abstract] ABSTRACT: Important regions of rRNA are rich in nucleotide modifications that can have strong effects on ribosome biogenesis and translation efficiency. Here, we examine the influence of pseudouridylation and 2′-O-methylation on translation accuracy in yeast, by deleting the corresponding guide snoRNAs. The regions analyzed were: the decoding centre (eight modifications), and two intersubunit bridge domains—the A-site finger and Helix 69 (six and five modifications). Results show that a number of modifications influence accuracy with effects ranging from 0.3- to 2.4-fold of wild-type activity. Blocking subsets of modifications, especially from the decoding region, impairs stop codon termination and reading frame maintenance. Unexpectedly, several Helix 69 mutants possess ribosomes with increased fidelity. Consistent with strong positional and synergistic effects is the finding that single deletions can have a more pronounced phenotype than multiple deficiencies in the same region. Altogether, the results demonstrate that rRNA modifications have significant roles in translation accuracy.
- [Show abstract] [Hide abstract] ABSTRACT: The mechanisms leading to non-lethality of nonsense mutations in essential genes are poorly understood. Here, we focus on the factors influencing viability of yeast cells bearing premature termination codons (PTCs) in the essential gene SUP45 encoding translation termination factor eRF1. Using a dual reporter system we compared readthrough efficiency of the natural termination codon of SUP45 gene, spontaneous sup45-n (nonsense) mutations, nonsense mutations obtained by site-directed mutagenesis (76Q --> TAA, 242R --> TGA, 317L --> TAG). The nonsense mutations in SUP45 gene were shown to be situated in moderate contexts for readthrough efficiency. We showed that readthrough efficiency of some of the mutations present in the sup45 mutants is not correlated with full-length Sup45 protein amount. This resulted from modification of both sup45 mRNA stability which varies 3-fold among sup45-n mutants and degradation rate of mutant Sup45 proteins. Our results demonstrate that some substitutions in the place of PTCs decrease Sup45 stability. The viability of sup45 nonsense mutants is therefore supported by diverse mechanisms that control the final amount of functional Sup45 in cells.
- [Show abstract] [Hide abstract] ABSTRACT: The large subunit rRNA in eukaryotes contains an unusually dense cluster of 8-10 pseudouridine (Psi) modifications located in a three-helix structure (H37-H39) implicated in several functions. This region is dominated by a long flexible helix (H38) known as the "A-site finger" (ASF). The ASF protrudes from the large subunit just above the A-site of tRNA binding, interacts with 5 S rRNA and tRNA, and through the terminal loop, forms a bridge (B1a) with the small subunit. In yeast, the three-helix domain contains 10 Psis and 6 are concentrated in the ASF helix (3 of the ASF Psis are conserved among eukaryotes). Here, we show by genetic depletion analysis that the Psis in the ASF helix and adjoining helices are not crucial for cell viability; however, their presence notably enhances ribosome fitness. Depleting different combinations of Psis suggest that the modification pattern is important and revealed that loss of multiple Psis negatively influences ribosome performance. The effects observed include slower cell growth (reduced rates up to 23% at 30 degrees C and 40-50% at 37 degrees C and 11 degrees C), reduced level of the large subunit (up to 17%), impaired polysome formation (appearance of half-mers), reduced translation activity (up to 20% at 30 degrees C and 25% at 11 degrees C), and increased sensitivity to ribosome-based drugs. The results indicate that the Psis in the three-helix region improve fitness of a eukaryotic ribosome.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
- [Show abstract] [Hide abstract] ABSTRACT: The yeast inheritable [URE3] element corresponds to a prion form of the nitrogen catabolism regulator Ure2p. We have isolated several orthologous URE2 genes in different yeast species: Saccharomyces paradoxus, S. uvarum, Kluyveromyces lactis, Candida albicans, and Schizosaccharomyces pombe. We show here by in silico analysis that the GST-like functional domain and the prion domain of the Ure2 proteins have diverged separately, the functional domain being more conserved through the evolution. The more extreme situation is found in the two S. pombe genes, in which the prion domain is absent. The functional analysis demonstrates that all the homologous genes except for the two S. pombe genes are able to complement the URE2 gene deletion in a S. cerevisiae strain. We show that in the two most closely related yeast species to S. cerevisiae, i.e., S. paradoxus and S. uvarum, the prion domains of the proteins have retained the capability to induce [URE3] in a S. cerevisiae strain. However, only the S. uvarum full-length Ure2p is able to behave as a prion. We also show that the prion inactivation mechanisms can be cross-transmitted between the S. cerevisiae and S. uvarum prions.
- [Show abstract] [Hide abstract] ABSTRACT: The aggregation of the two yeast proteins Sup35p and Ure2p is widely accepted as a model for explaining the prion propagation of the phenotypes [PSI+] and [URE3], respectively. Here, we demonstrate that the propagation of [URE3] cannot simply be the consequence of generating large aggregates of Ure2p, because such aggregation can be found in some conditions that are not related to the prion state of Ure2p. A comparison of [PSI+] and [URE3] aggregation demonstrates differences between these two prion mechanisms. Our findings lead us to propose a new unifying model for yeast prion propagation.
- [Show abstract] [Hide abstract] ABSTRACT: In the yeast Saccharomyces cerevisiae, the non-Mendelian inherited genetic element [URE3] behaves as a prion. A hypothesis has been put forward which states that [URE3] arises spontaneously from its cellular isoform Ure2p (the product of the URE2 gene), and propagates through interactions of the N-terminal domain of the protein, thus leading to its aggregation and loss of function. In the present study, various N- and C-terminal deletion mutants of Ure2p were constructed and their cross-interactions were tested in vitro and in vivo using affinity binding and a two-hybrid analysis. We show that the self-interaction of the protein is mediated by at least two domains, corresponding to the first third of the protein (the so-called prion-forming domain) and the C-terminal catalytic domain.
- [Show abstract] [Hide abstract] ABSTRACT: In 1993, a pilot project for the functional analysis of newly discovered open reading frames, presumably coding for proteins, from yeast chromosome III was launched by the European Community. In the frame of this programme, we have developed a large-scale screening for the identification of gene/protein functions via systematic phenotypic analysis. To this end, some 80 haploid mutant yeast strains were constructed, each carrying a targeted deletion of a single gene obtained by HIS3 or TRP1 transplacement in the W303 background and a panel of some 100 growth conditions was established, ranging from growth substrates, stress to, predominantly, specific inhibitors and drugs acting on various cellular processes. Furthermore, co-segregation of the targeted deletion and the observed phenotype(s) in meiotic products has been verified. The experimental procedure, using microtiter plates for phenotypic analysis of yeast mutants, can be applied on a large scale, either on solid or in liquid media. Since the minimal working unit of one 96-well microtiter plate allows the simultaneous analysis of at least 60 mutant strains, hundreds of strains can be handled in parallel. The high number of monotropic and pleiotropic phenotypes (62%) obtained, together with the acquired practical experience, have shown this approach to be simple, inexpensive and reproducible. It provides a useful tool for the yeast community for the systematic search of biochemical and physiological functions of unknown genes accounting for about a half of the 6000 genes of the complete yeast genome.
- [Show abstract] [Hide abstract] ABSTRACT: During the functional analysis of open reading frames (ORFs) identified during the sequencing of chromosome III of Saccharomyces cerevisiae, the previously uncharacterized ORF YCL031C (now designated RRP7) was deleted. RRP7 is essential for cell viability, and a conditional null allele was therefore constructed, by placing its expression under the control of a regulated GAL promoter. Genetic depletion of Rrp7p inhibited the pre-rRNA processing steps that lead to the production of the 20S pre-rRNA, resulting in reduced synthesis of the 18S rRNA and a reduced ratio of 40S to 60S ribosomal subunits. A screen for multicopy suppressors of the lethality of the GAL::rrp7 allele isolated the two genes encoding a previously unidentified ribosomal protein (r-protein) that is highly homologous to the rat r-protein S27. When present in multiple copies, either gene can suppress the lethality of an RRP7 deletion mutation and can partially restore the ribosomal subunit ratio in Rrp7p-depleted cells. Deletion of both r-protein genes is lethal; deletion of either single gene has an effect on pre-rRNA processing similar to that of Rrp7p depletion. We believe that Rrp7p is required for correct assembly of rpS27 into the preribosomal particle, with the inhibition of pre-rRNA processing appearing as a consequence of this defect.
- [Show abstract] [Hide abstract] ABSTRACT: The sequence of the genome of Saccharomyces cerevisiae was recently determined. As well as all the informations concerning the structure of the chromosomes the scientific community had to deal with the discovery of dozens of new open reading frames (ORFs) of unknown function. The study of these ORFs requires the development of simple procedures that can be used on a large scale. In the framework of a European Pilot Project we have described a new approach for deleting ORFs. This method is based on transformation with a polymerase chain reaction product but is limited by the use of a strain deleted for the auxotropic marker. We present here the construction of a new recipient strain that lacks the TRP1 region and that allows a high efficiency of gene deletion.
Université Paris-Sud 11
Orsay, Île-de-France, France
- Institut de Génétique et Microbiologie (IGMORS)
Pierre and Marie Curie University - Paris 6Lutetia Parisorum, Île-de-France, France
French National Centre for Scientific Research
Lutetia Parisorum, Île-de-France, France
- Centre of Molecular Genetics
Center for Molecular GeneticsGif, Île-de-France, France