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Ribosome biogenesis and nucleocytoplasmic transport
Rentrée doctorale Ribosome biogenesis and nucleocytoplasmic transport Ecm1 - a new pre-60S nuclear export factor in S. cerevisiae Yanhua YAO Supervisor: Micheline FROMONT-RACINE Unité de Génétique des Interactions Macromoléculaires URA CNRS2171, Institut Pasteur 1
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Why study ribosome biogenesis?
Essential Pathway Very expensive for the cell (2000 ribosomes/min.) Highly regulated (stress, cell cycle ….) Highly conserved across the evolution 2
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Ribosome biogenesis in eucaryotes
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Since 2000...... Tandem Affinity Purification * Identification of
Ribosomal proteins Erb1 Noc2, Mak5 Nop2, Sda1, Puf6 Dbp7, Nop7, Yhr197w, Nog1 Arx1, Nug1, Dbp9 Fpr3, Nop12, Ssf1 Fpr4, Nog2 Ycr072c, Ytm1, Mak11, Cic1 Mak16, Rlp7, Rpf2 Nop1, Rrp1 Vps75, Brx1, Nsa2, Nop16 Tif6 Yor252w Pre-ribosomal proteins Rlp24-TAP Nog1-TAP Mtr4 * Tandem Affinity Purification Rigaut et al. 1999, Nat. Biotechnol. Identification of 45 new pre-60S factors (Mass Spectrometry) Saveanu et al. 2003, M. C. B.
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Pre-60S factors’ Recycling
? Lebreton et al. 2006, J.C.B. 5
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How the pre-60S particles are exported?
Question: How the pre-60S particles are exported?
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Pre-60S factors’ Recycling
Lebreton et al. 2006, J.C.B. 7 7
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Genetic Interactions Network
M X Z Y B C A (Decourty et al. 2008, P.N.A.S.) Genetic Interactions Network ARX1 Nuclear Pore Complexe ECM1 Not essential (Gavin et al., 2002, Nature) Synthetic Lethality with nug1-1 and mtr2-33 (Bassler et al., 2001, Mol. Cell)
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Validation of genetic interactions
Double mutant: Slow growth or sick growth Conclusion 1: When the NPC (nuclear pore complex) is compromised, Ecm1 is essential. 9 9
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The depletion of Ecm1 affecte 60S biognesis
Rpl25-GFP WT ecm1∆ pom34∆ PGAL1-ECM1 pom34∆ Conclusion 2: When the NPC is affected, Ecm1 becomes essential for pre-60S nuclear export. 10 10
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Ecm1- a pre-60S factor 1 Kap121 2 Kap108 3 Nog1 4 Fpr4 5 Nog2 Rpl Conclusion 3: Ecm1 is conditionally associated with pre-60S particles. 11
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Two-hybid Yeast Screen
Binding domain on X Y (Brent and Ptashne 1985; Fields and Song, 1989) Activating domain Two-hybid Yeast Screen X: Ecm1 Y: Nup192 Conclusion 4: Ecm1 binds physically to NPC.
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adapted from Bradatsch et al., 2007 Gadal et al., 2001 Ho et al., 2000
Hung et al., 2008 Yao et al., 2007 Lorsque le nucleopore est affecté, Ecm1 devient essentiel. Son absence provoque la rétentions des particules pré-60S dans le noyau. Il est lié directement à pré-60S et NPC. Tous sont similaire de la fonction de Arx1. Donc Ecm1 est un 4ème récepteur d’export de particules pré-60S. 13 13 13
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Ecm1 overlaps with other export pathway
Conclusion 5: Ecm1, Arx1 and Mex67 show subtle functional interactions involved in the same export process, but each protein has additional important cellular roles.
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Conclusions and Perspectives:
A novel additional pre-60S nuclear export factor. Ecm1 But… adapted from Bradatsch et al., 2007 Lorsque le nucleopore est affecté, Ecm1 devient essentiel. Son absence provoque la rétentions des particules pré-60S dans le noyau. Il est lié directement à pré-60S et NPC. Tous sont similaire de la fonction de Arx1. Donc Ecm1 est un 4ème récepteur d’export de particules pré-60S. Ecm1 and Arx1 in the same complexe ? How is pre-60S export regulated ? 15 15
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Acknowledgements: GIM’s Lab: Plateforme protéomique: Pascal Lenormand
Micheline Fromont-Racine Alain Jacquier Cosmin Saveanu Laurence Decourty Emilie Demoinet Helen Neil Odile Labouise Christophe Malabat
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Combining gene deletions (synthetic lethality, epistasy, suppression):
- neutral - aggravating - alleviating N M X Z Y B C A
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Genetic Interactions Mapping (GIM)
Competitive growth of double mutants (1 tube) Diploids obtention Meiosis MATα Query mutation ∆X MAT A 5,000 systematic deletion strains MAT A MATA Genetic Interactions Mapping (GIM)
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barcodes PCR amplification
potential aggravating int. ~ duplicated spots Yeast BarCode 13.5k microarray (SLRI, Toronto) potential alleviating int. -> log2(Query/Reference) for ~5 000 double mut. Query population Reference population
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Principe du double-hybride
Brent and Ptashne 1985; Fields and Song (1989) on Domaine de liaison a l’ADN Domaine D'activation off Y X off Y X on 20
