A complex molecular machinery to specifically incorporate selenocysteine

into proteins important for health and disease

Laurence Wurth

Supervisor: Christine AllmangGroup Alain Krol

Architecture et Réactivité de l’ARN Strasbourg/France

Tuapse 2008

Se34

78.96C

COO -

+H3N CH2SeHH

Selenocysteine

Essential micronutrient

Selenoproteins

Selenium

Selenocysteine : the 21st amino acid

Eukaryotic selenoproteins

15 kDa DI1 DI2 DI3 GPx1 GPx2 GPx3 GPx4 GPx6 MsrA SelH SelI SelK SelM SelN SelO SelP MsrB SelS SelT SelU SelV SelW SPS2 Trx1 Trx2 Trx3

From Driscoll et al. 2004

(?)

??????

????(?)

? = proteins of unknown function

Glutathione peroxidases

Deiodinases

Thioredoxin reductases

Functions of selenoproteins

Protection against free radicals

Development

Male Fertility

Muscle development

Unknown function for most of the selenoproteins

Thyroid function

Diseases linked to deficient selenoproteins

Rigid spine muscular dystrophy (deficient SelN)

cancer

Thyroid diseases

Importance of selenoproteins in human health Growing interest in understanding how selenoproteins

are produced in the cell

Keshan disease (cardiomyopathy)

Male infertility

Early rigidity of the spine

Muscle weakness and atrophy

Respiratory failure

Protein synthesis in eukaryotic cells

DNA=Genetic information

mRNA

Coding region

Non-coding region

ribosome

messenger RNA

protein

Initiation

7mG(ppp)G AAAAAAAAUG stop

UGAUAAUAG

Initiationfactor

aa

Protein synthesis in eukaryotic cells

Coding region

Non-coding region

ribosome

messenger RNA

protein

Initiation

7mG(ppp)G AAAAAAAAUG stop

UGAUAAUAG

Protein synthesis in eukaryotic cells

Coding region

Non-coding region

ribosome

messenger RNA

protein

7mG(ppp)G AAAAAAAAUG stop

UGAUAAUAG

Elongfactor

aa

Elongation

Protein synthesis in eukaryotic cells

Coding region

Non-coding region

ribosome

messenger RNA

protein

7mG(ppp)G AAAAAAAAUG stop

UGAUAAUAG

Elongation

Protein synthesis in eukaryotic cells

Coding region

Non-coding region

ribosome

messenger RNA

protein

7mG(ppp)G AAAAAAAAUG stop

UGAUAAUAG

Elongation

Protein synthesis in eukaryotic cells

Coding region

Non-coding region

ribosome

messenger RNA

protein

7mG(ppp)G AAAAAAAAUG stop

UGAUAAUAG

Elongation

Protein synthesis in eukaryotic cells

Termination

Coding region

Non-coding region

ribosome

messenger RNA

protein

7mG(ppp)G AAAAAAAAUG stop

UGAUAAUAG

Protein synthesis in eukaryotic cells

Termination

Coding region

Non-coding region

ribosome

messenger RNA

protein

7mG(ppp)G AAAAAAAAUG stop

UGAUAAUAG

UGA=canonical stop =Sec codon if reprogrammed

How is selenocysteine incorporated into selenoproteins ?

Which other factors are implicated?

selenoprotein7mG(ppp)G

AAAAAAA

AUG AUGUGA

EFSec

Sec

SBP2

?

stop

SECIS

mRNA

7mG(ppp)G

AAAAAAA

AUG

EFSec

UGA

SecSBP2

?

stop

SECISSBP2

Nucleus

AAAAAAA

Allmang C., Krol A., Biochimie (2006)de Jesus et al., Mol. Cell Biol. (2006)Papp LV. et al., Mol Cell Biol. (2006)

Cytoplasm

In which cellular compartment does SBP2 bind the SECIS RNA?

SBP2 colocalizes with selenoprotein mRNA in the nucleus

Does the assembly of the proteins on the mRNA take place in the nucleus?

Nucleus

Cytoplasm

Eukaryotic Cell

Confocal microscopy

SBP2-GFP: Green fluorescent protein

mRNA: probe linked to red fluorescent dye

colocalization

Yellow signal

SBP2-GFP: green mRNA GPX: red Combined: yellow DAPI: nucleus

hSBP2: a L7Ae RNA binding domain

SBP2 selenoprotein mRNAs h15.5kD/Snu13p C/D snoRNPs, snRNPsNhp2p H/ACA snoRNPsL30 rRNAs, mRNAsL7Ae sRNAs, rRNAs

L7Aemodule

L7Ae proteins bind RNAs that adopt the same structure

U4 snRNA L30e mRNA sRNA SECIS RNA

15.5K L30e L7Ae SBP2

SBP2: RNA binding domain of the L7Ae family of proteins

SECIS RNA: same structure as other small RNAs implicated in other mechanisms in the cell

Aim : understanding the general

assembly mechanismof the RNA-protein complexes

from the L7Ae family

EFsecsec

?

RNA-protein complex

3’ UTR5’

SBP2

3’ UTR5’

SBP2

Mature RNA-protein complex

??

Identification of a new factor associated to the L7Ae proteins

Nufip, what is its function?

human

Nufip

human

L7Ae

Nufip

Nufip: NUclear FMRPInteracting Protein

SBP2

Identification of a new factor associated to the L7Ae proteins

human

Nufip

human

L7Ae

Nufip

Nufip, what is its function?

Nufip: NUclear FMRPInteracting Protein

7mG(ppp)G AUG

EFSec

UGA

SecSBP2

?

stop

SECISSBP2

Nufip colocalizes with selenoprotein mRNA in the nucleus

Confocal microscopy

Nufip-GFP: Green fluorescent protein

mRNA: probe linked to red fluorescent dye

colocalization

Yellow signalNucleus

Cytoplasm

Eukaryotic Cell

Nufip-GFP: green mRNA GPX: red Combined: yellow DAPI: nucleus

Nufip acts as an adaptor to bring other factors to RNA-protein complexes

Nufip acts as an adaptor to bring other factors to RNA-protein complexes

Nufip

3’ UTR5’

L7Ae/SBP2

Other factors

Other factors

3’ UTR5’

L7Ae/SBP2

Nufip can tether L7Ae proteins to otherwise non interacting proteins

Does Nufip create a link to other biogenesis factors ?

Nufip interacts with a chaperone complex

Human R2TP complex

Hsp90 is a chaperone and the R2TP proteins are its adaptorsChaperones help proteins to fold correctly

SBP2

AAAAAAmRNA

Nufip

In vivo function of Hsp90 and its co-chaperones

The role of Hsp90 and the R2TP proteins

SBP2 GFP-15.5K

GFP-hNhp2

GA GA

control

GA blocs ATPase siteHsp90

Hsp90 is inhibited by Geldanamycin (GA) :

Prevents foldingof client proteins

ATPase site

Hsp90 Hsp90Functioninhibited

The role of Hsp90 and the R2TP proteins

Hsp90 may control proper L7Ae protein folding during the assemblyof RNA-protein complexes (RNPs)

SBP2 GFP-15.5K

GFP-hNhp2

GA GA

control

SBP2 and other L7Ae proteins are client proteins of Hsp90

GA blocs ATPase siteHsp90

Hsp90 is inhibited by Geldanamycin (GA) :

Prevents foldingof client proteins

ATPase site

Hsp90 Hsp90Functioninhibited

RNPsnoRNPsnRNPselenoprotein mRNP

A conserved assembly and folding machinery

L7Ae/SBP2

Boulon*, Marmier-Gourrier*, Pradet-Balade*, Wurth* et al. *Equal contributionJ.Cell.Biol. (2008)

RNPsnoRNPsnRNPselenoprotein mRNP

Nufip adaptor protein

A conserved assembly and folding machinery

L7Ae/SBP2

Boulon*, Marmier-Gourrier*, Pradet-Balade*, Wurth* et al. *Equal contributionJ.Cell.Biol. (2008)

coreproteins

Nufip

R2TPCo-chaperones of Hsp90folding machinery

RNPsnoRNPsnRNPselenoprotein mRNP

Nufip adaptor protein

A conserved assembly and folding machinery

L7Ae/SBP2

Boulon*, Marmier-Gourrier*, Pradet-Balade*, Wurth* et al. *Equal contributionJ.Cell.Biol. (2008)

coreproteins

Nufip

R2TPCo-chaperones of Hsp90folding machinery

RNPsnoRNPsnRNPselenoprotein mRNP

Nufip adaptor protein

A conserved assembly and folding machinery

L7Ae/SBP2

Boulon*, Marmier-Gourrier*, Pradet-Balade*, Wurth* et al. *Equal contributionJ.Cell.Biol. (2008)

coreproteins

NufipNufip

Laurence WurthAnne Schweigert Christine AllmangAlain Krol

Institut de Génétique Moléculaire,Montpellier Edouard Bertrand

Université Henri Poincaré, NancyChristiane BranlantBruno Charpentier

Architecture et Réactivité de l’ARNInstitut de Biologie Moléculaire et Cellulaire,Strasbourg

QuickTime™ et undécompresseur TIFF (non compressé)

sont requis pour visionner cette image.