A complex molecular machinery to specifically incorporate selenocysteine into proteins important for...
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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
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