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1. p53 activation during ribosome biogenesis regulates normal erythroid differentiation

Author

François Morlé, Stéphane Giraudier, Eric Soler, Naomi Taylor, Charlotte Andrieu-Soler, Olivier Hermine, Patrick Mayeux, Célia Floquet, Rose Ann Padua, Frédérique Verdier, Sarah Ducamp, Michaela Fontenay, Mohammad Salma, Elisabeth M. Cramer-Borde, Ismael Boussaid, Anna Raimbault, Isabelle Hatin, Diane d'Allard, Amandine Houvert, Narla Mohandas, Boris Guyot, Emilie-Fleur Gautier, Sandrina Kinet, Marjorie Leduc, Pierre-Emmanuel Gleizes, Jean-Jacques Diaz, Salomé Le Goff, François Guillonneau, Nathalie Montel-Lehry, Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Cochin (IC UM3 (UMR 8104 / U1016)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de Génétique Moléculaire de Montpellier (IGMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM), Plateforme protéomique 3P5 [Institut Cochin] (3P5), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Génomique, Structure et Traduction (GST), Département Biologie des Génomes (DBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Excellence : Biogenèse et pathologies du globule rouge (Labex Gr-Ex), Université Sorbonne Paris Cité (USPC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Hématopoïèse normale et pathologique : émergence, environnement et recherche translationnelle [Paris] ((UMR_S1131 / U1131)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Institut NeuroMyoGène (INMG), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), New York Blood Center, Hôpital Cochin [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Procédés Alimentaires et Microbiologiques (PAM), Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement, ANR-18-IDEX-0001,Université de Paris,Université de Paris(2018), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], Immunology, Ribosome biogenesis, Biology, Biochemistry, Ribosome, Mice, 03 medical and health sciences, 0302 clinical medicine, Erythroid Cells, Downregulation and upregulation, Transcription (biology), Ribosomal protein, hemic and lymphatic diseases, Animals, Humans, Erythropoiesis, Gene, ComputingMilieux_MISCELLANEOUS, Organelle Biogenesis, RNA, Cell Differentiation, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, Hematology, Hematopoietic Stem Cells, Cell biology, 030104 developmental biology, 030220 oncology & carcinogenesis, Organelle biogenesis, Tumor Suppressor Protein p53, Ribosomes
Abstract

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q− syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle–arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.

Published
2021
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2. Integrated analyses of translatome and proteome identify the rules of translation selectivity in RPS14-deficient cells

Author

Michaela Fontenay, Pierre-Julien Viailly, Bertrand Cosson, Patrick Mayeux, Anna Raimbault, Dina Al Dulaimi, Emilie-Fleur Gautier, Barbara Burroni, Isabelle Dusanter-Fourt, Ismael Boussaid, Salomé Le Goff, Célia Floquet, Isabelle Hatin, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Génomique, Structure et Traduction (GST), Département Biologie des Génomes (DBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)

Subjects
Ribosomal Proteins, 5q- syndrome, Proteome, [SDV]Life Sciences [q-bio], Red Cells, Biology, Ribosomopathies, medicine.disease_cause, Ribosome, Article, 03 medical and health sciences, 0302 clinical medicine, Ribosomal protein, hemic and lymphatic diseases, medicine, Coding region, Humans, Diamond-Blackman, Anemia, Macrocytic, Gene, 030304 developmental biology, Anemia, Diamond-Blackfan, 0303 health sciences, Mutation, Translation (biology), GATA1, Hematology, Cell biology, 030220 oncology & carcinogenesis, Codon usage bias, Myelodysplastic Syndromes, Ribosomes
Abstract

In ribosomopathies, the Diamond-Blackfan anemia (DBA) or 5q- syndrome, ribosomal protein (RP) genes are affected by mutation or deletion, resulting in bone marrow erythroid hypoplasia. Unbalanced production of ribosomal subunits leading to a limited ribosome cellular content regulates translation at the expense of the master erythroid transcription factor GATA1. In RPS14-deficient cells mimicking 5q- syndrome erythroid defects, we show that the transcript length, codon bias of the coding sequence (CDS) and 3’UTR (untranslated region) structure are the key determinants of translation. In these cells, short transcripts with a structured 3’UTR and high codon adaptation index (CAI) showed a decreased translation efficiency. Quantitative analysis of the whole proteome confirmed that the post-transcriptional changes depended on the transcript characteristics that governed the translation efficiency in conditions of low ribosome availability. In addition, proteins involved in normal erythroid differentiation share most determinants of translation selectivity. Our findings thus indicate that impaired erythroid maturation due to 5q- syndrome may proceed from a translational selectivity at the expense of the erythroid differentiation program, and suggest that an interplay between the CDS and UTR may regulate mRNA translation.

Published
2020
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3. Divergent effects of translation termination factor eRF3A and nonsense-mediated mRNA decay factor UPF1 on the expression of uORF carrying mRNAs and ribosome protein genes

Author

Affaf Aliouat, Pauline François, Vérène Stierlé, Olivier Jean-Jean, Pierre Bertin, Samia Salhi, Isabelle Hatin, Olivier Namy, Adaptation Biologique et Vieillissement = Biological Adaptation and Ageing (B2A), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Département de Mathématiques et Applications - ENS Paris (DMA), Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Centre recherche en CardioVasculaire et Nutrition (C2VN), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Paris Seine (IBPS), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), École normale supérieure - Paris (ENS Paris)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Génomique, Structure et Traduction (GST), Département Biologie des Génomes (DBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), ANR-17-CE12-0024,Rescue_ribosome,Voies de sauvetage pour les ribosomes non recyclés(2017), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), and SERRE, Marie-Claude

Subjects
Ribosomal Proteins, eRF3, [SDV]Life Sciences [q-bio], Nonsense-mediated decay, MRNA Decay, nonsense-mediated mRNA decay, Biology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Ribosome, Open Reading Frames, 03 medical and health sciences, 0302 clinical medicine, RNA, Messenger, Molecular Biology, Gene, Translation termination, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, ribosome protein genes, GSPT1, RNA, translation termination, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, Nonsense Mediated mRNA Decay, Cell biology, [SDV] Life Sciences [q-bio], Gene Expression Regulation, 030220 oncology & carcinogenesis, UPF1, Trans-Activators, RNA Helicases, Peptide Termination Factors, Research Paper, uORF
Abstract

International audience; In addition to its role in translation termination, eRF3A has been implicated in the nonsense-mediated mRNA decay (NMD) pathway through its interaction with UPF1. NMD is a RNA quality control mechanism, which detects and degrades aberrant mRNAs as well as some normal transcripts including those that harbour upstream open reading frames in their 5' leader sequence. In this study, we used RNA-sequencing and ribosome profiling to perform a genome wide analysis of the effect of either eRF3A or UPF1 depletion in human cells. Our bioinformatics analyses allow to delineate the features of the transcripts controlled by eRF3A and UPF1 and to compare the effect of each of these factors on gene expression. We find that eRF3A and UPF1 have very different impacts on the human transcriptome, less than 250 transcripts being targeted by both factors. We show that eRF3A depletion globally derepresses the expression of mRNAs containing translated uORFs while UPF1 knockdown derepresses only the mRNAs harbouring uORFs with an AUG codon in an optimal context for translation initiation. Finally, we also find that eRF3A and UPF1 have opposite effects on ribosome protein gene expression. Together, our results provide important elements for understanding the impact of translation termination and NMD on the human transcriptome and reveal novel determinants of ribosome biogenesis regulation.

Published
2019
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4. Turnover of ribosome-associated transcripts from de novo ORFs produces gene-like characteristics available for de novo gene emergence in wild yeast populations

Author

Isabelle Gagnon-Arsenault, Christian R. Landry, Isabelle Hatin, Johan Hallin, Alexandre K. Dubé, Lou Nielly-Thibault, Eléonore Durand, Olivier Namy, Département de Biologie [Québec], Institut de Biologie Intégrative et des Systèmes [Québec] (IBIS)-Université Laval [Québec] (ULaval), Département de Biochimie, Microbiologie et Bioinformatique [Québec, Canada], Université Laval [Québec] (ULaval)-Pavillon Charles-Eugène-Marchand [Québec, Canada] (CHM), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Génomique, Structure et Traduction (GST), Département Biologie des Génomes (DBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), This work was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery grant and a Fonds de recherche Nature et Technologies Quebec (FRQNT) Team Grant (2015-PR-181807) to C.R.L. C.R.L. holds the Canada Research Chair in Evolutionary Cell and Systems Biology., Pavillon Charles-Eugène-Marchand [Québec, Canada] (CHM)-Université Laval [Québec] (ULaval), and Namy, Olivier

Subjects
Transcription, Genetic, Biology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Ribosome, Open Reading Frames, 03 medical and health sciences, 0302 clinical medicine, Intergenic region, Gene Expression Regulation, Fungal, Yeasts, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], [SDV.BID.EVO] Life Sciences [q-bio]/Biodiversity/Populations and Evolution [q-bio.PE], Genetics, Protein biosynthesis, Saccharomyces paradoxus, ORFS, Gene, Genetics (clinical), 030304 developmental biology, 0303 health sciences, Reporter gene, Research, [SDV.BID.EVO]Life Sciences [q-bio]/Biodiversity/Populations and Evolution [q-bio.PE], [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, biology.organism_classification, Biological Evolution, Open reading frame, Protein Biosynthesis, [SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Ribosomes, 030217 neurology & neurosurgery
Abstract

Little is known about the rate of emergence of de novo genes, what their initial properties are, and how they spread in populations. We examined wild yeast populations (Saccharomyces paradoxus) to characterize the diversity and turnover of intergenic ORFs over short evolutionary timescales. We find that hundreds of intergenic ORFs show translation signatures similar to canonical genes, and we experimentally confirmed the translation of many of these ORFs in laboratory conditions using a reporter assay. Compared with canonical genes, intergenic ORFs have lower translation efficiency, which could imply a lack of optimization for translation or a mechanism to reduce their production cost. Translated intergenic ORFs also tend to have sequence properties that are generally close to those of random intergenic sequences. However, some of the very recent translated intergenic ORFs, which appeared

Published
2019
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5. The high turnover of ribosome-associated transcripts from de novo ORFs produces gene-like characteristics available for de novo gene emergence in wild yeast populations

Author

Éléonore Durand, Isabelle Gagnon-Arsenault, Johan Hallin, Isabelle Hatin, Alexandre K Dubé, Lou Nielly-Thibaut, Olivier Namy, and Christian R Landry

Subjects
Genetics, 0303 health sciences, Mutation rate, Biology, biology.organism_classification, Genome, 03 medical and health sciences, 0302 clinical medicine, Intergenic region, Mutation (genetic algorithm), Gene duplication, Saccharomyces paradoxus, ORFS, Gene, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract

Little is known about the rate of emergence of genes de novo, how they spread in populations and what their initial properties are. We examined wild Saccharomyces paradoxus populations to characterize the diversity and turnover of intergenic ORFs over short evolutionary time-scales. We identified ~34,000 intergenic ORFs per individual genome for a total of ~64,000 orthogroups, which resulted from an estimated turnover rate relatively smaller than the rate of gene duplication in yeast. Hundreds of intergenic ORFs show translation signatures, similar to canonical genes, but lower translation efficiency, which could reduce their potential production cost or simply reflect a lack of optimization. Translated intergenic ORFs tend to display low expression levels with sequence properties that are on average closer to expectations based on intergenic sequences. However, some predicted de novo polypeptides with gene-like properties emerged from ancient as well as recent birth events, illustrating that the raw material for functional innovations may appear even over short evolutionary time-scales. Our results suggest that variation in the mutation rate along the genome impacts the turnover of random polypeptides, which may in turn influence their early evolutionary trajectory. Whereas low mutation rate regions allow more time for random intergenic ORFs to evolve and become functional before being lost, mutation hotspots allow for the rapid exploration of the molecular landscape, thereby increasing the probability to acquire a polypeptide with immediate gene-like properties and thus functional potential.

Published
2018
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6. Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli

Author

Jacob, Beal, Trac Haddock Angellii, Markus, Gershater, Kim de Mora, Meagan, Lizarazo, Jim, Hollenhorst, Randy, Rettberg, Philipp, Demling, Rene, Hanke, Michae, Osthegel, Anna, Schechtel, Suresh, Sudarsan, Arne, Zimmermann, Bartosz, Gabryelczyk, Martina, Ikonen, Minnamari, Salmela, Muradıye, Acar, Muhammed Fatih Aktas, Furkan, Bestepe, Furkan Sacit Ceylan, Sadık, Cigdem, Mikail, Dohan, Mustafa, Elitok, Mehmet, Gunduz, Esra, Gunduz, Omer Faruk Hatipoglu, Turan, Kaya, Orhan, Sayin, Safa, Tapan, Osman Faruk Tereci, Abdullah, Uçar, Mustafa, Yilmaz, Jeffrey, Barrick, Alex, Gutierrez, Dennis, Mishler, Jordan, Monk, Kate, Mortensen, Nathan, Shin, Ella, Watkins, Yintong, Chen, Yuji, Jin, Yuanjie, Shi, Haoqian Myelin Zhang, Bruno, Ono, Ieda Maria Martinez Paino, Lais, Ribovski, Ivan, Silva, Danilo Keiji Zampronio, Nils, Birkholz, Rudiger Frederik Busche, Oliver, Konzock, Steffen, Lippold, Carsten, Ludwig, Melanie, Philippi, Lukas, Platz, Christian, Sigismund, Susanne, Weber, Maren, Wehrs, Niels, Werchau, Anna, Wronska, Zen Zen Yen, Yash, Agarwal, Evan, Appleton, Douglas, Densmore, Ariela, Esmurria, Kathleen, Lewis, Alan, Pacheco, Marcel, Bruchez, Danielle, Peters, Cheryl, Telmer, Lena, Wang, Silvia Canas Duarte, Daniel Giraldo Perez, Camilo Gomez Garzon, Jorge Madrid Wolff, Nathaly Marin Medina, Valentina, Mazzanti, Laura Rodriguez Forero, Eitan, Scher, Robin, Dowell, Samantha, O’Hara, Cloe Simone Pogoda, Kendra, Shattuck, Ali, Altintas, Anne Pihl Bali, Rasmus, Bech, Anne, Egholm, Anne Sofie Laerke Hansen, Kristian, Jensen, Kristian Barreth Karlsen, Caroline, Mosbech, Sophia, Belkhelfa, Noemie, Berenger, Romain, Bodinier, Cecile, Jacry, Laura Matabishi Bibi, Pierre, Parutto, Julie, Zaworski, Andries de Vries, Freek de Wijs, Rick, Elbert, Lisa, Hielkema, Chandhuru, Jagadeesan, Bayu, Jayawardhana, Oscar, Kuipers, Anna, Lauxen, Thomas, Meijer, Sandra, Mous, Renske van Raaphorst, Aakanksha, Saraf, Otto, Schepers, Oscar, Smits, Jan Willem Veening, Ruud Detert Oude Weme, Lianne, Wieske, Catherine, Ainsworth, Xenia Spencer Milnes, Alejandro, Gómezávila, Eddie Cano Gamez, Ana Laura Torres Huerta, Carlos Alejandro Meza Ramirez, Philipp, Popp, Jara, Radeck, Anna, Sommer, Xiangkai, Li, Qi, Wu, Hongxia, Zhao, Ruixue, Zhao, Irem, Bastuzel, Yasemin, Ceyhan, Mayda, Gursel, Burak, Kizil, Ilkem, Kumru, Yasemin, Kuvvet, Helin, Tercan, Seniz, Yuksel, Luiza, Niyazmetova, Timothy, Ang, Lucas, Black, Ciaran, Kelly, George, Wadhams, Clovis, Basier, Urszula, Czerwinska, Cindy Suci Ananda, Muhammad Al Azhar, Adelia, Elviantari, Maya, Fitriana, Arief Budi Witarto, Yuliant, Jia Fangxing, Qingfeng, Hou, Wan, Pei, Chen, Rifei, Wang, Rong, Huang, Wei, Zhang, Yushan, Jianguo, He, Dengwen, Lai, Pai, Li, Jianheng, Liu, Chunyang, Ni, Qianbin, Zhang, Cinthya, Cadenas, Zardain Canabal, Eduardo J., Claudia Nallely Alonso Cantu, Mercedes Alejandra Vazquez Cantu, Eduardo Cepeda Canedo, Cesar Miguel Valdez Cordova, Jose Alberto de la Paz Espinosa, Carlos Enrique Alavez Garcia, Ana Laura Navarro Heredia, Adriana, Hernandez, Sebastian Valdivieso Jauregui, Eduardo Ramirez Montiel, Eduardo Serna Morales, Yamile Minerva Castellanos Morales, Omar Alonso Cantu Pena, Ramirez Rodríguez, Eduardo A., Elizabeth Vallejo Trejo, Jesus Gilberto Rodriguez Ceja, Jesus Eduardo Martinez Hernandez, Mario Alberto Pena Hernandez, Enrique Amaya Perez, Rebeca Paola Torres Ramirez, Cla, J., Martin, Hanzel, Sarah Mohand Said, Shihab, Sawar, Dylan, Siriwardena, Alex, Tzahristos, Nils, Anlind, Martin, Friberg, Erik, Gullberg, Stephanie, Herman, Dallin, Christensen, Sara, Gertsch, Cody, Maxfield, Charles, Miller, Ryan, Putman, Christine, Bauerl, Estelles Lopez, Lucia T., Estefania Huet Trujillo, Marta Vazquez Vilar, Marlène Sophie Birk, Nico, Claassens, Walter de Koster, Rik van Rosmalen, Wen Ying Wu, Sian, Davies, Dan, Goss, William, Rostain, Chelsey, Tye, Waqar, Yousaf, Natalie, Farny, Chloe, Lajeunesse, Alex, Turland, Chen, An, Jielin, Chen, Yahong, Chen, Zehua, Che, Baishan, Fang, Xiaotong, Fu, Xifeng, Guo, Yue, Jiang, Yiying, Lei, Jianqiao, Li, Zhe, Li, Chang, Liu, Weibing, Liu, Yang, Li, Yizhu, Lv, Qingyu, Ruan, Yue, Su, Chun, Tang, Yushen, Wang, Fan, Wu, Xiaoshan, Yan, Ruihua, Zhang, Tangduo, Zhang, Farren, Isaacs, Ariel Leyva Hernandez, Natalie, Ma, Stephanie, Mao, Yamini, Naidu, Tuukka, Miinalainen, Marion Aruann, Daniel Calendini, Yoann Chabert, Gael Chambonnier, Myriam, Choukour, Ella de Gaulejac, Camille, Houy, Axel, Levier, Loreen, Logger, Sebastien, Nin, Valerie, Prima, Sturgis, James N., Beibei, Fang, Sadik, Cigdem, Abdullah, Ucar, Alejandro, Gutierrez, Revanth, Poondla, Sanjana, Reddy, Tyler, Rocha, Natalie, Schulte, Devin, Wehle, Marta Eva Jackowski, Sean Ross Craig, Ariana Mirzarafie Ahi, Elliott, Parris, Luba, Prout, Barbara, Steijl, Rachel, Wellman, Zhao, Fan, Zhang, Jing, Yang, Wei, Yang, Yuanzhan, Wen, Zhaosen, Evan, Appletion, Jeffrey, Chen, Abha, Patil, Shaheer, Priracha, Kate, Ryan, Nick, Salvador, John, Viola, Boralli, Camila Maria S., Camila Barbosa Bramorski, Juliana Cancino Bernardi, Ana Laura de Lima, Paula Maria Pincela Lins, Cristiane Casonato Melo, Deborah Cezar Mendonca, Thiago, Mosqueiro, Everton, Silva, Graziele, Vasconcelos, Ruchi, Asthana, Donna, Lee, Michelle, Yu, Peter, Choi, Effie, Lau, Kenneth, Lau, Oscar, Ying, Brandon, Malone, Paul, Young, Aidan, Ceney, Dakota, Hawthorne, Sharon, Lian, Sam, Mellentine, Dylan, Miller, Barbara Castro Moreira, Christie, Peebles, Olivia, Smith, Kevin, Walsh, Allison, Zimont, Michael, Brasino, Michael, Donovan, Hannah, Young, Jan, Bejvl, Daniel, Georgiev, Hynek, Kasl, Katerina Pechotova, Vaclav Pelisek, Anna, Sosnova, Pavel, Zach, Anthony, Ciesla, Benjamin, Hoover, Elliott, Chapman, Jon Marles Wright, Vicky, Moynihan, Liusaidh, Owen, Brooke Rothschild Mancinelli, Emilie, Cuillery, Joseph, Heng, Vincent, Jacquot, Paola, Malsot, Rocco, Meli, Cyril, Pulver, Ari, Sarfatis, Loic, Steiner, Victor, Steininger, Nina van Tiel, Gregoire, Thouvenin, Axel, Uran, Lisa, Baumgartner, Anna, Fomitcheva, Daniel, Gerngross, Verena, Jagger, Michael, Meier, Anja, Michel, Jasmine, Bird, Bradley, Brown, Todd, Burlington, Daniel, Herring, Joseph, Slack, Georgina, Westwood, Emilia, Wojcik, Julian, Bender, Julia, Donauer, Ramona, Emig, Rabea, Jesser, Julika, Neumann, Lara, Stuhn, Takema, Hasegawa, Tomoya, Kozakai, Haruka, Maruyama, Sean, Colloms, Charlotte, Flynn, Vilija, Lomeikaite, James, Provan, Kang, Ning, Shuyan, Tang, Guozhao, Wu, Yunjun, Yang, Zhi, Zeng, Zhan, Yi, Pan, Chu, Jun, Li, Keji, Yan, Athale, Chaitanya A., Swapnil, Bodkhe, Manasi, Gangan, Harsh, Gakhare, Yash, Jawale, Snehal, Kadam, Prachiti, Moghe, Gayatri, Mundhe, Neha, Khetan, Ira, Phadke, Prashant, Uniyal, Siddhesh, Zadey, Ines, Cottignie, Eline, Deprez, Astrid, Deryckere, Jasper, Janssens, Frederik, Jonnaert, Katarzyna, Malczewska, Thomas, Pak, Johan, Robben, Ovia Margaret Thirukkumaran, Vincent Van Deuren, Laurens, Vandebroek, Laura Van Hese, Laetitia Van Wonterghem, Leen, Verschooten, Moritz, Wolter, Joss, Auty, Richard, Badge, Liam, Crawford, Raymond, Dalgleish, Amy, Evans, Cameron, Grundy, Charlie, Kruczko, Payal, Karia, Graeme, Glaister, Rhys, Hakstol, Seme, Mate, Karin, Otero, Dustin, Smith, Jeff, Tingley, Hans Joachim Wieden, Haotian, Wang, Ningning, Yao, Matthias, Franz, Anna, Knoerlein, Nicolas, Koutsoubelis, Loechner, Anne C., Max, Mundt, Alexandra, Richter, Oliver, Schauer, Marjorie, Buss, Sivateja, Tangirala, Brian, Teague, Tianyi, Huang, Xinhao, Song, Yibing, Wei, Zhaoran, Zhang, Longzhi, Cao, Cheng Li, Kang Yang, Zhiqin, Chen, Yuxing, Fang, Libo, Sun, Weiyi, Wang, Yang, Yang, David, Adams, Joshua, Colls, Joshua, Timmons, David, Urick, Julia Anna Adrian, Madina, Akan, Youssef, Chahibi, Rahmi, Lale, Typhaine Le Doujet, Marit Vaagen Roee, Altynay, Abdirakhmanova, Askarbek, Orakov, Azhar, Zhailauova, Jinyang, Liang, Yu, Ma, Qikai, Qin, Yetian, Su, Ju Yeon Han, Raphaella, Hull, Wei Chung Kong, Li Chieh Lu, Duke, Quinton, Pauline, Aubert, Johan, Bourdarias, Olivier, Bugaud, Coralie Demon Chaine, Isabelle, Hatin, Ibtissam Kaid Slimane, Seong Koo Kang, Audrey, Moatti, Cheikh Fall Ndiaye, Mathilde, Ananos, Alexander, Arkhipenko, Valentin, Bailly, Jules, Caput, Javier, Castillo, Alma Chapet Batlle, Floriane, Cherrier, Claudia Demarta Gatsi, Deshmukh, Gopaul, Muriel, Gugger, Caroline, Lambert, Lucas, Krauss, Amelie, Vandendaele, Xiaojing, Li, Lin, Xiaomei, Luo Xunxun, Anders C. h. r. Hansen, Tina, Kronborg, Pettersen, Jens S., Charles, Calvet, Tyler Dae Devlin, Kosuke, Fujishima, Danny, Greenberg, Tina, Ju, Ryan, Kent, Daniel, Kunin, Erica, Lieberman, Griffin, Mccutcheon, Thai, Nguyen, Lynn, Rothschild, Shih, Joseph D., Jack, Takahashi, Kirsten, Thompson, Forrest, Tran, Daniel, Xiang, Felix, Richter, Yang, Xiaoran, Xiangyue, Hu, Changyuan, Deng, Shuyu, Hua, Yumeng, Li, Xinyu, Meng, Boxiang, Wang, Yingqi, Wang, Xuan, Wang, Zixuan, Xu, Jieyu, Yan, Ming, Yan, Yineng, Zhou, Edgar Alberto Alcalá Orozco, José Alberto Cristerna Bermúdez, Daniela Flores Gómez, José Ernesto Hernández Castañeda, Diana Clarisse Montaño Navarro, Juana Yessica PérezÁvila, María Fernanda Salazar Figueroa, María Fernanda Sánchez Arroyo, Oliva Angélica Sánchez Montesinos, Ángel Farid Rojas Cruz, Carlos Ramos Gutiérrez, Alonso Pérez Lona, Carlos Alejandro Meza Ramírez, Fernanda Sotomayor Olivares, Jorge Sebastián Rodríguez Iniesta, Juan Carlos Rueda Silva, Shotaro, Ayukawa, Takahiro, Kashiwagi, Daisuke, Kiga, Misa, Minegishi, Riku, Shinohara, Hiraku, Tokuma, Yuta, Yamazaki, Shuhei, Yasunishi, Erinn Sim Zixuan, Remsha, Afzal, Matthew, Carrigan, Barry, Moran, Marlena, Mucha, Arnas, Petrauskas, Stefan, Marsden, Michelle, Post, Anne, Rodenburg, Hector, Sanguesa, Marit van der Does, Erwin van Rijn, Max van’t Hof, Yeshi de Bruin, Hans de Ferrante, Elles, Elschot, Laura, Jacobs, Jan Willem Muller, Sjoerd, Nooijens, Femke, Vaassen, Cas van der Putten, Esther van Leeuwen, Laura van Smeden, Kwan Kwan Zhu, Kevin, Sabath, Katharina, Sporbeck, Nicolai von Kügelgen, Lisa, Wellinger, Stefanie, Braun, Jack, Ho, Yash, Mishra, Mariola, Sebastian, Lucas von Chamier, Ahsan, Fasih M., Satyadi, Megan A., Vivienne, Gunadhi, Phillip, Kyriakakis, Jenny, Lee, Walter, Thavarajah, Kimia, Abtahi, Robert, Hand, Chun Mun Loke, Adam, Wahab, Iowis, Zhu, Del Bianco, Cristina, Chizzolini, Fabio, Elisa, Godino, Lentini, Roberta, Mansy, Sheref Samir, Yeh Martin, Noel, Claudio Oss Pegorar, Alexander, Cook, Timothy, Kerns, Chad, Nielsen, Michael, Paskett, Alexander, Torgesen, Stephen, Lee, Ophir, Ospovat, Sikandar, Raza, Daniel, Shaykevich, Jarrod, Shilts, Barbora, Bajorinaite, Mykolas, Bendorius, Ieva, Rauluseviciute, Ieva, Savickyte, Sarunas, Tumas, William, Buchser, Elli, Cryan, Caroline, Golino, Andrew, Halleran, Taylor, Jacobs, Michael, Lefew, Joe, Maniaci, John, Marken, Margaret, Saha, Panya, Vij, Kayla, Desanty, Julie, Mazza, Raytheon BBN Technologies, iGEM Foundation, Synthace, Agilent Technology [Santa Clara], Raytheon BBN Technologies, Synthace, and Agilent provided support in the form of salaries for authors JB, MG, and JH, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the author contributions section, The authors wish to thank Sarah Munro and Marc Salit of NIST for help in designing this study. Consortium authors include all persons self-identified by contributing teams as deserving co-authorship credit. Contributors are listed alphabetically within team, and teams alphabetically and by year. Note that some persons may be credited as contributing in both years. Team names are given as identified in iGEM records: full details of each team’s institution and additional members may be found online in the iGEM Foundation archives at:http://year.igem.org/Team:name e.g.: full information on the 2015 ETH_Zurich team may be found at: http://2015.igem.org/Team:ETH_Zurich, BBN Technologies, IGEM Foundation, Synthace Ltd., Agilent Technologies, Department of Biotechnology and Chemical Technology, Helsinki Institute for Information Technology HIIT, ATOMS Turkiye, Boston University, Carnegie Mellon University, Technical University of Denmark, Ludwig Maximilian University of Munich, Middle East Technical University, Sumbawa University of Technology, Southern University of Science and Technology, Sun Yat-Sen University, Tecnológico de Monterrey, Universidad Autonoma de Nuevo Leon, University of Ottawa, Universitat Politècnica de València, Wageningen University and Research Centre, Worcester Polytechnic Institute, Xiamen University, University of Texas at Austin, Bielefeld University, Birkbeck University of London, USP-Brazil, City University of Hong Kong, Colorado State University, CU Boulder, Swiss Federal Institute of Technology Lausanne, Swiss Federal Institute of Technology Zurich, University of Exeter, Indian Institute of Science Education and Research, KU Leuven, Massachusetts Institute of Technology, Northeast Agricultural University, Nanjing Agricultural University, Norwegian University of Science and Technology, Ocean University of China, University of Southern Denmark, Shenzhen Middle School - SZMS 15, Tokyo Institute of Technology, Trinity College Dublin, Delft University of Technology, Eindhoven University of Technology, Tuebingen, University of California Los Angeles, University of California San Diego, University of Maryland, University of Trento, Vanderbilt University, College of William and Mary, Department of Bioproducts and Biosystems, Aalto-yliopisto, Aalto University, Jones, D Dafydd, Discrete Technology and Production Automation, and ​Robotics and image-guided minimally-invasive surgery (ROBOTICS)

Subjects
0106 biological sciences, 0301 basic medicine, green fluorescent protein, Laboratory Proficiency Testing, Transcription, Genetic, International Genetically Engineered Machine, [SDV]Life Sciences [q-bio], lcsh:Medicine, Protein Engineering, 01 natural sciences, Infographics, Synthetic biology, genetics, lcsh:Science, Promoter Regions, Genetic, Macromolecular Engineering, transcription initiation, Measurement, Multidisciplinary, Chemistry, Strain (biology), gene expression regulation, good laboratory practice, Research Assessment, Fluorescence, Reproducibility, 3. Good health, Bioassays and Physiological Analysis, Engineering and Technology, Educational Status, Synthetic Biology, Genetic Engineering, Transcription, Graphs, Research Article, Biotechnology, Transcriptional Activation, Computer and Information Sciences, General Science & Technology, Green Fluorescent Proteins, Bioengineering, Computational biology, iGEM Interlab Study Contributors, Research and Analysis Methods, Promoter Regions, 03 medical and health sciences, promoter region, Genetic, 010608 biotechnology, Escherichia coli, ta215, business.industry, Data Visualization, lcsh:R, Fluorescence Competition, genetic transcription, DNA structure, Reproducibility of Results, Biology and Life Sciences, protein engineering, 030104 developmental biology, Good Health and Well Being, 7 INGENIERÍA Y TECNOLOGÍA, Synthetic Bioengineering, People and Places, lcsh:Q, Population Groupings, biosynthesis, business, metabolism, Undergraduates
Abstract

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices. Copyright: © 2016 Beal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Published
2016
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7. A role for the universal Kae1/Qri7/YgjD (COG0533) family in tRNA modification

Author

Christopher Deutsch, Basma El Yacoubi, Isabelle Hatin, Valérie de Crécy-Lagard, Jean-Pierre Rousset, Alexey G. Murzin, Dirk Iwata-Reuyl, and Tamer Kahveci

Subjects
Comparative genomics, Genetics, 0303 health sciences, TRNA modification, General Immunology and Microbiology, Protein family, General Neuroscience, Mutant, Saccharomyces cerevisiae, Biology, biology.organism_classification, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, 0302 clinical medicine, Telomere Homeostasis, Start codon, Transcriptional regulation, Molecular Biology, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract

The YgjD/Kae1 family (COG0533) has been on the top-10 list of universally conserved proteins of unknown function for over 5 years. It has been linked to DNA maintenance in bacteria and mitochondria and transcription regulation and telomere homeostasis in eukaryotes, but its actual function has never been found. Based on a comparative genomic and structural analysis, we predicted this family was involved in the biosynthesis of N6-threonylcarbamoyl adenosine, a universal modification found at position 37 of tRNAs decoding ANN codons. This was confirmed as a yeast mutant lacking Kae1 is devoid of t6A. t6A− strains were also used to reveal that t6A has a critical role in initiation codon restriction to AUG and in restricting frameshifting at tandem ANN codons. We also showed that YaeZ, a YgjD paralog, is required for YgjD function in vivo in bacteria. This work lays the foundation for understanding the pleiotropic role of this universal protein family.

Published
2011
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8. Molecular dissection of translation termination mechanism identifies two new critical regions in eRF1

Author

Céline Fabret, Olivier Namy, Jean-Pierre Rousset, Isabelle Hatin, Institut de génétique et microbiologie [Orsay] (IGM), and Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Silent mutation, Models, Molecular, MESH: Mutation, Saccharomyces cerevisiae Proteins, MESH: Amino Acid Sequence, Saccharomyces cerevisiae, Biology, medicine.disease_cause, MESH: Phenotype, Ribosome, Conserved sequence, 03 medical and health sciences, MESH: Protein Structure, Tertiary, MESH: Saccharomyces cerevisiae Proteins, Genetics, medicine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Conserved Sequence, 030304 developmental biology, 0303 health sciences, Mutation, MESH: Genetic Complementation Test, MESH: Conserved Sequence, 030302 biochemistry & molecular biology, Peptide Termination Factors, Genetic Complementation Test, Peptide Chain Termination, Translational, MESH: Saccharomyces cerevisiae, Stop codon, Protein Structure, Tertiary, Open reading frame, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Phenotype, MESH: Gene Deletion, Proofreading, RNA, MESH: Peptide Chain Termination, Translational, MESH: Models, Molecular, MESH: Peptide Termination Factors, Gene Deletion
Abstract

International audience; Translation termination in eukaryotes is completed by two interacting factors eRF1 and eRF3. In Saccharomyces cerevisiae, these proteins are encoded by the genes SUP45 and SUP35, respectively. The eRF1 protein interacts directly with the stop codon at the ribosomal A-site, whereas eRF3-a GTPase protein-probably acts as a proofreading factor, coupling stop codon recognition to polypeptide chain release. We performed random PCR mutagenesis of SUP45 and screened the library for mutations resulting in increased eRF1 activity. These mutations led to the identification of two new pockets in domain 1 (P1 and P2) involved in the regulation of eRF1 activity. Furthermore, we identified novel mutations located in domains 2 and 3, which confer stop codon specificity to eRF1. Our findings are consistent with the model of a closed-active conformation of eRF1 and shed light on two new functional regions of the protein.

Published
2009
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9. Translation Analysis at the Genome Scale by Ribosome Profiling

Author

Olivier Namy, Rachel Legendre, Agnès Baudin-Baillieu, Isabelle Hatin, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule ( I2BC ), and Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 )

Subjects
0301 basic medicine, MESH : Protein Biosynthesis, MESH : RNA, Messenger, [SDV]Life Sciences [q-bio], 5.8S ribosomal RNA, Computational biology, Biology, Ribosome profiling, Deep sequencing, 03 medical and health sciences, Ribo-seq, Translational regulation, MESH: Genome, MESH: High-Throughput Nucleotide Sequencing, MESH: RNA, Messenger, MESH : Ribosomes, Genetics, [ SDV ] Life Sciences [q-bio], Recoding, Shine-Dalgarno sequence, Translation (biology), Ribosomal binding site, 030104 developmental biology, Translation regulation, MESH: Protein Biosynthesis, MESH : Polyribosomes, MESH : Genome, Eukaryotic Ribosome, MESH: Polyribosomes, MESH: Ribosomes, MESH : High-Throughput Nucleotide Sequencing
Abstract

International audience; 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.

Published
2016
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10. Fine-Tuning of Translation Termination Efficiency inSaccharomyces cerevisiaeInvolves Two Factors in Close Proximity to the Exit Tunnel of the Ribosome

Author

Wayne A. Decatur, Isabelle Hatin, Jean-Pierre Rousset, Olivier Namy, Céline Fabret, Institut de génétique et microbiologie [Orsay] (IGM), and Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Saccharomyces cerevisiae Proteins, Carboxy-Lyases, Macromolecular Substances, Termination factor, Genes, Fungal, Saccharomyces cerevisiae, Gene Expression, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Investigations, Ribosome, 03 medical and health sciences, Peptide Elongation Factor 1, Genes, Reporter, Genetics, RNA, Small Nucleolar, HSP70 Heat-Shock Proteins, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, DNA, Fungal, Gene, 030304 developmental biology, 0303 health sciences, Messenger RNA, Base Sequence, biology, 030302 biochemistry & molecular biology, RNA, Fungal, Peptide Chain Termination, Translational, Ribosomal RNA, biology.organism_classification, Stop codon, Mutagenesis, Codon, Terminator, Ribosomes, Genetic screen
Abstract

In eukaryotes, release factors 1 and 3 (eRF1 and eRF3) are recruited to promote translation termination when a stop codon on the mRNA enters at the ribosomal A-site. However, their overexpression increases termination efficiency only moderately, suggesting that other factors might be involved in the termination process. To determine such unknown components, we performed a genetic screen in Saccharomyces cerevisiae that identified genes increasing termination efficiency when overexpressed. For this purpose, we constructed a dedicated reporter strain in which a leaky stop codon is inserted into the chromosomal copy of the ade2 gene. Twenty-five antisuppressor candidates were identified and characterized for their impact on readthrough. Among them, SSB1 and snR18, two factors close to the exit tunnel of the ribosome, directed the strongest antisuppression effects when overexpressed, showing that they may be involved in fine-tuning of the translation termination level.

Published
2007
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11. RiboTools: a Galaxy toolbox for qualitative ribosome profiling analysis

Author

Olivier Namy, Isabelle Hatin, Agnès Baudin-Baillieu, Rachel Legendre, Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), ANR-2011-BSV6-011-02,Blanc SVSE 6,Rôle du NSD/NGD: identification des gènes cibles et des facteurs impliqués dans ces mécanismes chez Saccharomyces cerevisiae ( 2011 ), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), and ANR-11-BSV6-0011,NGD-NSD,Rôle du NSD/NGD: identification des gènes cibles et des facteurs impliqués dans ces mécanismes chez Saccharomyces cerevisiae(2011)

Subjects
Statistics and Probability, animal structures, Prions, [SDV]Life Sciences [q-bio], Computational biology, Saccharomyces cerevisiae, Biology, Bioinformatics, Biochemistry, 03 medical and health sciences, 0302 clinical medicine, Qualitative analysis, Translational regulation, Databases, Genetic, Humans, Ribosome profiling, Molecular Biology, 030304 developmental biology, 0303 health sciences, [ SDV ] Life Sciences [q-bio], Gene Expression Profiling, Computational Biology, High-Throughput Nucleotide Sequencing, Toolbox, Galaxy, Stop codon, Computer Science Applications, Computational Mathematics, Computational Theory and Mathematics, Gene Expression Regulation, Protein Biosynthesis, Codon, Terminator, Genome, Fungal, Ribosomes, 030217 neurology & neurosurgery, Software
Abstract

Motivation: 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. Availability and implementation: 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. Contact: olivier.namy@igmors.u-psud.fr Supplementary Information: Supplementary data are available from Bioinformatics online.

Published
2014
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12. A Viable Hypomorphic Allele of the Essential IMP3 Gene Reveals Novel Protein Functions in Saccharomyces cerevisiae

Author

Sylvie Hermann-Le Denmat, Marta Kwapisz, Jean-Pierre Rousset, Olivier Namy, Céline Fabret, Antonin Morillon, Isabelle Hatin, Bruno Cosnier, Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Dynamique de l'information génétique : bases fondamentales et cancer (DIG CANCER), and Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Sorbonne Université (SU)

Subjects
DNA Repair, Ribosome biogenesis, Yeast and Fungal Models, Ribosome, Nucleic Acids, Gene Expression Regulation, Fungal, Molecular Cell Biology, Frameshift Mutation, Genetics, 0303 health sciences, Multidisciplinary, biology, Protein translation, 030302 biochemistry & molecular biology, Telomere, Stop codon, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Medicine, Genome, Fungal, Research Article, Ribosomal Proteins, Saccharomyces cerevisiae Proteins, Science, Saccharomyces cerevisiae, Frameshift mutation, 03 medical and health sciences, Model Organisms, Ribosomal protein, Genetic Mutation, Two-Hybrid System Techniques, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Gene, Biology, Alleles, 030304 developmental biology, Models, Genetic, Mutation Types, Ribosomal RNA, biology.organism_classification, Protein Structure, Tertiary, RNA processing, Polyribosomes, Mutation, RNA, Gene expression, Gene Function, Ribosomes, DNA Damage
Abstract

International audience; In Saccharomyces cerevisiae, the essential IMP3 gene encodes a component of the SSU processome, a large ribonucleoprotein complex required for processing of small ribosomal subunit RNA precursors. Mutation of the IMP3 termination codon to a sense codon resulted in a viable mutant allele producing a C-terminal elongated form of the Imp3 protein. A strain expressing the mutant allele displayed ribosome biogenesis defects equivalent to IMP3 depletion. This hypomorphic allele represented a unique opportunity to investigate and better understand the Imp3p functions. We demonstrated that the +1 frameshifting was increased in the mutant strain. Further characterizations revealed involvement of the Imp3 protein in DNA repair and telomere length control, pointing to a functional relationship between both pathways and ribosome biogenesis.

Published
2011
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13. Lack of pseudouridine 38/39 in the anticodon arm of yeast cytoplasmic tRNA decreases in vivo recoding efficiency

Author

François Lecointe, George Simos, Olivier Namy, Henri Grosjean, Jean-Pierre Rousset, Isabelle Hatin, Laboratoire d'Enzymologie et Biochimie Structurales (LEBS), Centre National de la Recherche Scientifique (CNRS), Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), University of Thessaly, CNRS, Association pour la Recherche sur le Cancer [Contract 5297], Ministère de l’Education Nationale de la Recherche et de la Technologie, Association pour la Recherche sur le Cancer [Contract 9873], Association Française Contre les Myopathies [Contract 7757], Ministère de la Recherche et de l’Enseignement Supérieur (predoctoral fellowship), 'Association pour la Recherche sur le Cancer' (predoctoral fellowship), Greek-French Collaboration Program 'PLATON', and Grosjean, Henri

Subjects
Cytoplasm, [SDV]Life Sciences [q-bio], Saccharomyces cerevisiae, Biology, Biochemistry, Ribosome, Pseudouridine, Frameshift mutation, 03 medical and health sciences, chemistry.chemical_compound, RNA, Transfer, Mutant protein, Anticodon, Molecular Biology, 030304 developmental biology, Genetics, 0303 health sciences, 030302 biochemistry & molecular biology, Frameshifting, Ribosomal, Translation (biology), Cell Biology, Stop codon, Nonsense suppressor, chemistry, Protein Biosynthesis, Transfer RNA, Ribosomes
Abstract

International audience; Many different modified nucleotides are found in naturally occurring tRNA, especially in the anticodon region. Their importance for the efficiency of the translational process begins to be well documented. Here we have analyzed the in vivo effect of deleting genes coding for yeast tRNA-modifying enzymes, namely Pus1p, Pus3p, Pus4p, or Trm4p, on termination readthrough and +1 frameshift events. To this end, we have transformed each of the yeast deletion strains with a lacZ-luc dual-reporter vector harboring selected programmed recoding sites. We have found that only deletion of the PUS3 gene, encoding the enzyme that introduces pseudouridines at position 38 or 39 in tRNA, has an effect on the efficiency of the translation process. In this mutant, we have observed a reduced readthrough efficiency of each stop codon by natural nonsense suppressor tRNAs. This effect is solely due to the absence of pseudouridine 38 or 39 in tRNA because the inactive mutant protein Pus3[D151A]p did not restore the level of natural readthrough. Our results also show that absence of pseudouridine 39 in the slippery tRNA(UAG)(Leu) reduces +1 frameshift efficiency. Therefore, the presence of pseudouridine 38 or 39 in the tRNA anticodon arm enhances misreading of certain codons by natural nonsense tRNAs as well as promotes frameshifting on slippery sequences in yeast.

Published
2002
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14. Statistical Analysis of Readthrough Levels for Nonsense Mutations in Mammalian Cells Reveals a Major Determinant of Response to Gentamicin

Author

Célia Floquet, Isabelle Hatin, Laure Bidou, Jean-Pierre Rousset, Institut de génétique et microbiologie [Orsay] (IGM), and Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Cancer Research, lcsh:QH426-470, viruses, Nonsense mutation, Gene Expression, Context (language use), Biology, Molecular Genetics, Cytosine, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, RNA, Transfer, Genetics, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Nucleotide, Amino Acids, Uracil, Molecular Biology, Cells, Cultured, Genetics (clinical), Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, Protein Synthesis Inhibitors, chemistry.chemical_classification, 0303 health sciences, Statistics, fungi, Aminoglycoside, RNA, Human Genetics, Peptide Chain Termination, Translational, Stop codon, 3. Good health, lcsh:Genetics, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, chemistry, Codon, Nonsense, Transfer RNA, Codon, Terminator, Gentamicins, Mathematics, 030217 neurology & neurosurgery, Research Article
Abstract

The efficiency of translation termination depends on the nature of the stop codon and the surrounding nucleotides. Some molecules, such as aminoglycoside antibiotics (gentamicin), decrease termination efficiency and are currently being evaluated for diseases caused by premature termination codons. However, the readthrough response to treatment is highly variable and little is known about the rules governing readthrough level and response to aminoglycosides. In this study, we carried out in-depth statistical analysis on a very large set of nonsense mutations to decipher the elements of nucleotide context responsible for modulating readthrough levels and gentamicin response. We quantified readthrough for 66 sequences containing a stop codon, in the presence and absence of gentamicin, in cultured mammalian cells. We demonstrated that the efficiency of readthrough after treatment is determined by the complex interplay between the stop codon and a larger sequence context. There was a strong positive correlation between basal and induced readthrough levels, and a weak negative correlation between basal readthrough level and gentamicin response (i.e. the factor of increase from basal to induced readthrough levels). The identity of the stop codon did not affect the response to gentamicin treatment. In agreement with a previous report, we confirm that the presence of a cytosine in +4 position promotes higher basal and gentamicin-induced readthrough than other nucleotides. We highlight for the first time that the presence of a uracil residue immediately upstream from the stop codon is a major determinant of the response to gentamicin. Moreover, this effect was mediated by the nucleotide itself, rather than by the amino-acid or tRNA corresponding to the −1 codon. Finally, we point out that a uracil at this position associated with a cytosine at +4 results in an optimal gentamicin-induced readthrough, which is the therapeutically relevant variable., Author Summary Nonsense mutations are single-nucleotide variations within the coding sequence of a gene that result in a premature termination codon. The presence of such mutations leads to the synthesis of a truncated protein unable to fulfill its normal function. Over the last ten years, treatment strategies have emerged based on the use of molecules, such as aminoglycoside antibiotics (gentamicin) that facilitate the readthrough of premature termination codons, thus restoring the synthesis of a full-length protein. Such strategies have been tested for various genetic diseases, including Duchenne muscular dystrophy and cystic fibrosis. The readthrough level depends on the nature of the stop codon and the surrounding nucleotide context, but little was known of the rules governing readthrough level and response to aminoglycosides. In this study, we use a large set of nonsense mutations for an in-depth statistical analysis designed to decipher the element of the nucleotide context responsible for modulating readthrough levels. We analyse the impact of the six nucleotides upstream and downstream from the stop codon. We demonstrate that the presence of a uracil residue immediately upstream the stop codon is associated with a stronger response to gentamicin treatment than the presence of any of the other three nucleotides.

Published
2012
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15. Global translational impacts of the loss of the tRNA modification t(6)A in yeast

Author

Valérie de Crécy-Lagard, Alvaro Glavic, Agnès Baudin-Baillieu, Patrick C. Thiaville, Rachel Legendre, Isabelle Hatin, Diego Rojas-Benítez, Olivier Namy, Guilhem Chalancon, University of Florida Genetics Institute, University of Florida [Gainesville], Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Department of Microbiology and Cell Science, Genetics and Genomics Graduate Program, FONDAP Center for Genome Regulation, Laboratory of Molecular Biology, MRC, University of Florida Genetics Institute (UFGI), University of Florida [Gainesville] (UF), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), and FONDAP Center for Genome Regulation (CGR)

Subjects
0301 basic medicine, TRNA modification, [SDV]Life Sciences [q-bio], Mutant, translation, modified nucleosides, Biology, Biochemistry, Genetics and Molecular Biology (miscellaneous), Microbiology, Applied Microbiology and Biotechnology, 03 medical and health sciences, Eukaryotic translation, Virology, Genetics, Ribosome profiling, Molecular Biology, Gene, lcsh:QH301-705.5, tRNA, ribosome profiling, [ SDV ] Life Sciences [q-bio], Translation (biology), Cell Biology, 030104 developmental biology, lcsh:Biology (General), t6A, Transfer RNA, Parasitology, Protein folding
Abstract

International audience; 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.

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16. A novel mutant of the Sup35 protein of Saccharomyces cerevisiae defective in translation termination and in GTPase activity still supports cell viability

Author

Jean Pierre Rousset, Sylvie Gillet, Pierre Maréchal, Sergey Lekomtsev, Bruno Cosnier, Isabelle Hatin, Céline Fabret, Institut de génétique et microbiologie [Orsay] (IGM), and Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Saccharomyces cerevisiae Proteins, lcsh:QH426-470, Prions, Termination factor, Saccharomyces cerevisiae, Biology, GTP Phosphohydrolases, 03 medical and health sciences, 0302 clinical medicine, Protein structure, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Phosphorylation, lcsh:QH573-671, Protein kinase A, Molecular Biology, 030304 developmental biology, 0303 health sciences, lcsh:Cytology, Peptide Termination Factors, Translational readthrough, Peptide Chain Termination, Translational, Cyclic AMP-Dependent Protein Kinases, Stop codon, Protein Structure, Tertiary, lcsh:Genetics, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Biochemistry, Mutation, Sup45p, Release factor, Ribosomes, 030217 neurology & neurosurgery, Protein Binding, Research Article
Abstract

Background When a stop codon is located in the ribosomal A-site, the termination complex promotes release of the polypeptide and dissociation of the 80S ribosome. In eukaryotes two proteins eRF1 and eRF3 play a crucial function in the termination process. The essential GTPase Sup35p, the eRF3 release factor of Saccharomyces cerevisiae is highly conserved. In particular, we observed that all eRF3 homologs share a potential phosphorylation site at threonine 341, suggesting a functional role for this residue. The goal of this study was to determine whether this residue is actually phosphorylated in yeast and if it is involved in the termination activity of the protein. Results We detected no phosphorylation of the Sup35 protein in vivo. However, we show that it is phosphorylated by the cAMP-dependent protein kinase A on T341 in vitro. T341 was mutated to either alanine or to aspartic acid to assess the role of this residue in the activity of the protein. Both mutant proteins showed a large decrease of GTPase activity and a reduced interaction with eRF1/Sup45p. This was correlated with an increase of translational readthrough in cells carrying the mutant alleles. We also show that this residue is involved in functional interaction between the N- and C-domains of the protein. Conclusion Our results point to a new critical residue involved in the translation termination activity of Sup35 and in functional interaction between the N- and C-domains of the protein. They also raise interesting questions about the relation between GTPase activity of Sup35 and its essential function in yeast.

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