Prsentation PowerPoint

1
A complex molecular machinery to specifically
incorporate selenocysteine into proteins
important for health and disease
Laurence Wurth Supervisor Christine
Allmang Group Alain Krol Architecture et
Réactivité de lARN Strasbourg/France Tuapse
2008
2
Selenocysteine the 21st amino acid
Selenocysteine
Selenium
Selenoproteins
Essential micronutrient
3
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
(?) ? ? ? ? ? ? ? ? ? ? (?)
Deiodinases
Glutathione peroxidases
? proteins of unknown function
Thioredoxin reductases
From Driscoll et al. 2004
4
Functions of selenoproteins
Protection against free radicals
Development
Male Fertility
Thyroid function
Muscle development
Unknown function for most of the selenoproteins
5
Diseases linked to deficient selenoproteins
Male infertility
Thyroid diseases
Keshan disease (cardiomyopathy)
Rigid spine muscular dystrophy (deficient SelN)
cancer
Importance of selenoproteins in human health
Growing interest in understanding how
selenoproteins are produced in the cell
6
Protein synthesis in eukaryotic cells
Coding region
messenger RNA
Non-coding region
ribosome
protein
7
Protein synthesis in eukaryotic cells
Initiation
Coding region
messenger RNA
Non-coding region
ribosome
protein
8
Protein synthesis in eukaryotic cells
Elongation
Coding region
messenger RNA
Non-coding region
ribosome
protein
9
Protein synthesis in eukaryotic cells
Elongation
Coding region
messenger RNA
Non-coding region
ribosome
protein
10
Protein synthesis in eukaryotic cells
Elongation
Coding region
messenger RNA
Non-coding region
ribosome
protein
11
Protein synthesis in eukaryotic cells
Elongation
Coding region
messenger RNA
Non-coding region
ribosome
protein
12
Protein synthesis in eukaryotic cells
UGA UAA UAG
Termination
Coding region
messenger RNA
Non-coding region
ribosome
protein
13
Protein synthesis in eukaryotic cells
UGA UAA UAG
Termination
Coding region
messenger RNA
Non-coding region
ribosome
protein
14
How is selenocysteine incorporated into
selenoproteins ?
AAAAAAA
?
SECIS
SBP2
stop
AUG
7mG(ppp)G
AUG
UGA
mRNA
UGAcanonical stop Sec codon if
reprogrammed
Which other factors are implicated?
15
In which cellular compartment does SBP2 bind the
SECIS RNA?
Cytoplasm
AAAAAAA
7mG(ppp)G
Allmang C., Krol A., Biochimie (2006) de Jesus et
al., Mol. Cell Biol. (2006) Papp LV. et al., Mol
Cell Biol. (2006)
16
SBP2 colocalizes with selenoprotein mRNA in the
nucleus
Confocal microscopy
Does the assembly of the proteins on the mRNA
take place in the nucleus?
17
hSBP2 a L7Ae RNA binding domain
L7Ae module
18
L7Ae proteins bind RNAs that adopt the same
structure
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
19
sec
?
EFsec
?
?
SBP2
SBP2
Mature RNA-protein complex
RNA-protein complex
Aim understanding the general assembly
mechanism of the RNA-protein complexes from the
L7Ae family
20
Identification of a new factor associated to the
L7Ae proteins
human
human
SBP2
L7Ae
Nufip
Nufip
Nufip NUclear FMRP Interacting Protein
Nufip, what is its function?
21
Identification of a new factor associated to the
L7Ae proteins
human
human
SBP2
L7Ae
7mG(ppp)G
Nufip
Nufip
Nufip NUclear FMRP Interacting Protein
Nufip, what is its function?
22
Nufip colocalizes with selenoprotein mRNA in the
nucleus
Confocal microscopy
23
Nufip can tether L7Ae proteins to otherwise non
interacting proteins
Other factors
Nufip
Other factors
L7Ae/ SBP2
3 UTR
5
Nufip acts as an adaptor to bring other factors
to RNA-protein complexes
24
Does Nufip create a link to other biogenesis
factors ?
25
Nufip interacts with a chaperone complex
Human R2TP complex
Hsp90 is a chaperone and the R2TP proteins are
its adaptors Chaperones help proteins to fold
correctly
26
In vivo function of Hsp90 and its co-chaperones
27
The role of Hsp90 and the R2TP proteins
GA blocs ATPase site
Prevents folding of client proteins
Function inhibited
Hsp90
Hsp90
Hsp90
Hsp90 is inhibited by Geldanamycin (GA)
ATPase site
28
The role of Hsp90 and the R2TP proteins
GA blocs ATPase site
Prevents folding of client proteins
Function inhibited
Hsp90
Hsp90
Hsp90
Hsp90 is inhibited by Geldanamycin (GA)
ATPase site
GA
GA
SBP2
GFP-15.5K
control
GFP-hNhp2
SBP2 and other L7Ae proteins are client proteins
of Hsp90
Hsp90 may control proper L7Ae protein folding
during the assembly of RNA-protein complexes
(RNPs)
29
A conserved assembly and folding machinery
L7Ae/ SBP2
RNP snoRNP snRNP selenoprotein mRNP
Boulon, Marmier-Gourrier, Pradet-Balade,
Wurth et al. Equal contribution J.Cell.Biol.
(2008)
30
A conserved assembly and folding machinery
Nufip
core proteins
Nufip adaptor protein
L7Ae/ SBP2
RNP snoRNP snRNP selenoprotein mRNP
Boulon, Marmier-Gourrier, Pradet-Balade,
Wurth et al. Equal contribution J.Cell.Biol.
(2008)
31
A conserved assembly and folding machinery
R2TP Co-chaperones of Hsp90 folding machinery
Nufip
Nufip adaptor protein
core proteins
L7Ae/ SBP2
RNP snoRNP snRNP selenoprotein mRNP
Boulon, Marmier-Gourrier, Pradet-Balade,
Wurth et al. Equal contribution J.Cell.Biol.
(2008)
32
A conserved assembly and folding machinery
R2TP Co-chaperones of Hsp90 folding machinery
Nufip
Nufip
Nufip adaptor protein
core proteins
L7Ae/ SBP2
RNP snoRNP snRNP selenoprotein mRNP
Boulon, Marmier-Gourrier, Pradet-Balade,
Wurth et al. Equal contribution J.Cell.Biol.
(2008)
33

Institut de Génétique Moléculaire, Montpellier
Edouard Bertrand Université Henri Poincaré,
Nancy Christiane Branlant Bruno Charpentier
Architecture et Réactivité de lARN Institut de
Biologie Moléculaire et Cellulaire, Strasbourg
Laurence Wurth Anne Schweigert Christine
Allmang Alain Krol