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A complex molecular machinery to specifically incorporate selenocysteine into proteins important for health and disease Laurence Wurth Supervisor: Christine.

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Presentation on theme: "A complex molecular machinery to specifically incorporate selenocysteine into proteins important for health and disease Laurence Wurth Supervisor: Christine."— Presentation transcript:

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 l’ARN Strasbourg/France Tuapse 2008

2 Se 34 78.96 C COO - + H 3 N CH 2 SeH H Selenocysteine Essential micronutrient Selenoproteins Selenium Selenocysteine : the 21 st amino acid

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 From Driscoll et al. 2004 (?) ? (?) ? = proteins of unknown function Glutathione peroxidases Deiodinases Thioredoxin reductases

4 Functions of selenoproteins Protection against free radicals Development Male Fertility Muscle development Unknown function for most of the selenoproteins Thyroid function

5 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

6 Protein synthesis in eukaryotic cells DNA=Genetic information mRNA Coding region Non-coding region ribosome messenger RNA protein Initiation 7mG(ppp)G AAAAAAA AUG stop UGA UAA UAG Initiation factor aa

7 Protein synthesis in eukaryotic cells Coding region Non-coding region ribosome messenger RNA protein Initiation 7mG(ppp)G AAAAAAA AUG stop UGA UAA UAG

8 Protein synthesis in eukaryotic cells Coding region Non-coding region ribosome messenger RNA protein 7mG(ppp)G AAAAAAA AUG stop UGA UAA UAG Elong factor aa Elongation

9 Protein synthesis in eukaryotic cells Coding region Non-coding region ribosome messenger RNA protein 7mG(ppp)G AAAAAAA AUG stop UGA UAA UAG Elongation

10 Protein synthesis in eukaryotic cells Coding region Non-coding region ribosome messenger RNA protein 7mG(ppp)G AAAAAAA AUG stop UGA UAA UAG Elongation

11 Protein synthesis in eukaryotic cells Coding region Non-coding region ribosome messenger RNA protein 7mG(ppp)G AAAAAAA AUG stop UGA UAA UAG Elongation

12 Protein synthesis in eukaryotic cells Termination Coding region Non-coding region ribosome messenger RNA protein 7mG(ppp)G AAAAAAA AUG stop UGA UAA UAG

13 Protein synthesis in eukaryotic cells Termination Coding region Non-coding region ribosome messenger RNA protein 7mG(ppp)G AAAAAAA AUG stop UGA UAA UAG

14 UGA=canonical stop =Sec codon if reprogrammed How is selenocysteine incorporated into selenoproteins ? Which other factors are implicated? selenoprotein 7mG(ppp)G AAAAAAA AUG UGA EFSec Sec SBP2 ? stop SECIS mRNA

15 7mG(ppp)G AAAAAAA AUG EFSec UGA Sec SBP2 ? stop SECIS SBP2 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?

16 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: greenmRNA GPX: redCombined: yellow DAPI: nucleus

17 hSBP2: a L7Ae RNA binding domain SBP2selenoprotein mRNAs h15.5kD/Snu13p C/D snoRNPs, snRNPs Nhp2pH/ACA snoRNPs L30rRNAs, mRNAs L7AesRNAs, rRNAs L7Ae module

18 L7Ae proteins bind RNAs that adopt the same structure U4 snRNAL30e mRNAsRNASECIS RNA 15.5KL30eL7AeSBP2 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 Aim : understanding the general assembly mechanism of the RNA-protein complexes from the L7Ae family EFsec sec ? RNA-protein complex 3’ UTR5’ SBP2 3’ UTR5’ SBP2 Mature RNA-protein complex ? ?

20 Identification of a new factor associated to the L7Ae proteins Nufip, what is its function? human Nufip human L7Ae Nufip Nufip: NUclear FMRP Interacting Protein SBP2

21 Identification of a new factor associated to the L7Ae proteins human Nufip human L7Ae Nufip Nufip, what is its function? Nufip: NUclear FMRP Interacting Protein 7mG(ppp)G AUG EFSec UGA Sec SBP2 ? stop SECIS SBP2

22 Nufip colocalizes with selenoprotein mRNA in the nucleus Confocal microscopy Nufip-GFP: Green fluorescent protein mRNA: probe linked to red fluorescent dye colocalization Yellow signal Nucleus Cytoplasm Eukaryotic Cell Nufip-GFP: greenmRNA GPX: red Combined: yellow DAPI: nucleus

23 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

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 SBP2 AAAAAA mRNA Nufip

26 In vivo function of Hsp90 and its co-chaperones

27 The role of Hsp90 and the R2TP proteins SBP2 GFP-15.5K GFP-hNhp2 GA control GA blocs ATPase site Hsp90 Hsp90 is inhibited by Geldanamycin (GA) : Prevents folding of client proteins ATPase site Hsp90 Function inhibited

28 The role of Hsp90 and the R2TP proteins Hsp90 may control proper L7Ae protein folding during the assembly of RNA-protein complexes (RNPs) SBP2 GFP-15.5K GFP-hNhp2 GA control SBP2 and other L7Ae proteins are client proteins of Hsp90 GA blocs ATPase site Hsp90 Hsp90 is inhibited by Geldanamycin (GA) : Prevents folding of client proteins ATPase site Hsp90 Function inhibited

29 RNP snoRNP snRNP selenoprotein mRNP A conserved assembly and folding machinery L7Ae/ SBP2 Boulon*, Marmier-Gourrier*, Pradet-Balade*, Wurth* et al. *Equal contribution J.Cell.Biol. (2008)

30 RNP snoRNP snRNP selenoprotein mRNP Nufip adaptor protein A conserved assembly and folding machinery L7Ae/ SBP2 Boulon*, Marmier-Gourrier*, Pradet-Balade*, Wurth* et al. *Equal contribution J.Cell.Biol. (2008) core proteins Nufip

31 R2TP Co-chaperones of Hsp90 folding machinery RNP snoRNP snRNP selenoprotein mRNP Nufip adaptor protein A conserved assembly and folding machinery L7Ae/ SBP2 Boulon*, Marmier-Gourrier*, Pradet-Balade*, Wurth* et al. *Equal contribution J.Cell.Biol. (2008) core proteins Nufip

32 R2TP Co-chaperones of Hsp90 folding machinery RNP snoRNP snRNP selenoprotein mRNP Nufip adaptor protein A conserved assembly and folding machinery L7Ae/ SBP2 Boulon*, Marmier-Gourrier*, Pradet-Balade*, Wurth* et al. *Equal contribution J.Cell.Biol. (2008) core proteins Nufip

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

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