Ribosome biogenesis and nucleocytoplasmic transport

Ecm1 - a new pre-60S nuclear export factor

in S. cerevisiae

Unité de Génétique des Interactions MacromoléculairesURA CNRS2171, Institut Pasteur

Yanhua YAO

Supervisor: Micheline FROMONT-RACINE

Rentrée doctorale 20081015-17

Why study ribosome biogenesis?

• Essential Pathway • Very expensive for the cell (2000 ribosomes/min.)

• Highly regulated (stress, cell cycle ….)

• Highly conserved across the evolution

Ribosome biogenesis in eucaryotes

Identification of45 new pre-60S factors

(Mass Spectrometry)

Saveanu et al. 2003, M. C. B.

Ribosomal proteins

Erb1Noc2, Mak5Nop2, Sda1, Puf6Dbp7, Nop7, Yhr197w, Nog1Arx1, Nug1, Dbp9Fpr3, Nop12, Ssf1Fpr4, Nog2Ycr072c, Ytm1, Mak11, Cic1Mak16, Rlp7, Rpf2Nop1, Rrp1Vps75, Brx1, Nsa2, Nop16

Tif6

Yor252w

Pre-ribosomal proteins

Rlp24

-TAP

Nog1-TAP

Mtr4*

*

Since 2000......

Rigaut et al. 1999, Nat. Biotechnol.

Tandem Affinity Purification

Pre-60S factors’ Recycling

Lebreton et al. 2006, J.C.B.

?

Question:

How the pre-60S particles are exported?

Pre-60S factors’ Recycling

Lebreton et al. 2006, J.C.B.

•Not essential (Gavin et al., 2002, Nature)•Synthetic Lethality with nug1-1 and mtr2-33 (Bassler et al., 2001, Mol. Cell)

Genetic Interactions Network

ARX1

Nuclear Pore

ComplexeECM1

N

M

X

Z

YBCA

N

M

X

Z

YCA

(Decourty et al. 2008, P.N.A.S.)

B

Validation of genetic interactions

Double mutant: Slow growth or sick growth

Conclusion 1: When the NPC (nuclear pore complex) is compromised, Ecm1 is essential.

The depletion of Ecm1 affecte 60S biognesis

Conclusion 2: When the NPC is affected, Ecm1 becomes

essential for pre-60S nuclear export.

Rpl25-GFP

WT

ecm1∆

pom34∆

PGAL1-ECM1 pom34∆

Ecm1- a pre-60S factor

1 Kap1212 Kap1083 Nog14 Fpr45 Nog2Rpl

Conclusion 3: Ecm1 is conditionally associated with pre-60S particles.

Binding domain

onX

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.

adapted from Bradatsch et al., 2007

Gadal et al., 2001Ho et al., 2000

Bradatschet al., 2007Hung et al., 2008

Yao et al., 2007

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.

Ecm1

Conclusions and Perspectives:

A novel additional pre-60S nuclear export factor.

adapted from Bradatsch et al., 2007

• Ecm1 and Arx1 in the same complexe ?

• How is pre-60S export regulated ?

But…

Micheline Fromont-Racine

Alain Jacquier

Cosmin Saveanu

Laurence Decourty

Emilie Demoinet

Helen Neil

Odile Labouise

Christophe Malabat

Acknowledgements:

GIM’s Lab: Plateforme protéomique:

Pascal Lenormand

Combining gene deletions (synthetic lethality, epistasy, suppression):

- neutral

- aggravating

- alleviating

N

M

X

Z

YBCA

N

B

M

X

Z

YBCA

5,000 systematic deletion strains

MATαQuery mutation∆X

MAT A

MATAMAT A

Diploidsobtention Meiosis

Competitivegrowth of doublemutants (1 tube)

Genetic Interactions Mapping (GIM)

Query population

Reference population

barcodes PCR amplification

barcodes PCR amplification

potential aggravating int.

~ 26.000 duplicated spots Yeast BarCode 13.5kmicroarray (SLRI, Toronto)

potential alleviating int.

-> log2(Query/Reference) for ~5 000 double mut.

20

Principe du double-hybridePrincipe du double-hybride

Domaine deliaison a l’ADN

on

off

onXY

XY

off

Brent and Ptashne 1985; Fields and Song (1989)

Domaine

D'activation