What was the biological question addressed? What major claims to the authors make?

1. What was the biological question addressed? • What major claims to the authors make? • What are the major results that the authors obtained? • Did the experiments presented justify the claims made? • What major unanswered questions were raised by this work? • What experiments need to be done to answer such questions?

3. Background: Prokaryotic translation IF1=eIF1A IF2=eIF5B IF3=eIF1 Larson, B. et. al (2005) Microbiol Mol Biol Rev

4. Background: HCV • HCV is a flavivirus that contains an IRES in the 5’ UTR • HCV and HCV-like IRES only need a small sub set of initiation factors Lukavsky, PJ (2008) Virus Research

5. Background: HCV • The HCV IRES can bind to the 40S subunit • eIF3 and eIF2-GTP-Met-tRNAiMet are needed to form the 48S pre-initiation complex Lukavsky, PJ (2008) Virus Research

6. HCV after 48S complex formation • Would expect that: • eIF5 would hydrolyze GTP on eIF2-GTP-Met-tRNAiMet • eIF2-GDP would disassociate • 60S and 48S complex would release eIFs except eIF5B • GTP hydrolysis of eIF5B and 80S formation

7. 80S complex can be formed on HCV IRES without eIF2 • In vitro assembly reactions • Add in 40S, 60S, eIF2, Met-tRNAiMet, and GTP or GMPPNP • Eliminating eIF5 still get 80S formation • Eliminating eIF5B no 80S complexes formed

8. 80S complex can be formed on HCV IRES without eIF2 • In then presence of GMPPNP and all the factors still get 80S formation • Need eIF3, eIF5B and GTP or analog to get 80S formation • Hydrolysis of GTP is not required for 80S formation

9. eIF5B promotes tRNA binding to 40S-eIF3-HCV IRES complex • Two things could be going on • eIF5B is just involved in 80S complex formation • Like IF2, eIF5B could be involved in 80S complex formation and stabilizing Met-tRNAiMet • [S35]-Met-tRNAiMet,40S subunits, HCV IRES and eIF3 • eIF5B is required to place the Met-tRNAiMet

10. Elongation with 80S complexes assembled without eIF2 • 80S subunits form, but are they functional? • In vitro translation elongation reconstituted using HCV ORF with a stop codon added in • Used toeprinting assay to identify pretermination complexes

11. Elongation with 80S complexes assembled without eIF2 • Pretermination complexes formed with eIF3 and eIF5B • eIF2 is not needed for elongation

12. eIF2-independent mechanism operates in cell free system • eIF2-indpendent initiation is physiologically relevant • Using rabbit reticulocyte lysate with labeled HCV-IRES RNA • GMPPNP blocks eIF2 dependent pathway at 48S formation • dsRNA will activate PKR, which then phosphorylate eIF2 • Depleting eIF2 from the system U T

13. HCV IRES-driven translation in stressed transfected cells • Cells were transfected with a cap-dependent mRNA and a monocistronic HCV IRES mRNA • eIF2-a phosphorylation was induced • HCV IRES-mediated translation is relatively resistant to eIF2 inactivation

14. HCV IRES-driven translation in stressed transfected cells • HCV IRES-mediated translation is relatively resistant to eIF2 inactivation • Including IFNa, which is used as a treatment

15. Conclusion

16. Conclusion • The HCV IRES needs eIF3, eIF5B, and GTP form 80S complex • The HCV IRES does not need GTP hydrolysis or eIF2 • What does eIF3 do?

18. Background:ASH1 mRNA • ASH1 mRNA is localized to the bud tip during late anaphase • Translation of the mRNA is repressed until the mRNA is anchored at the bud tip • Puf6 and Khd1p are required for localization and translation repression Paquin, N. & Chartand (2008) Trends in Cell Biology

19. Background: Khd1p repression • Khd1p binds ASH1 mRNA and represses translation • ASH1 mRNA localizes to the bud tip • Khd1p gets phosphorylated by casein kinase I at the bud tip, and releases the mRNA • ASH1 mRNA then able to be translated Paquin, N., et. al (2007) Cell

20. Background: Puf6 • Puf6 binds a conserved UUGU sequence in the E3 domain of the ASH mRNA 3’UTR. • When bound Puf6 represses translation of the ASH1 mRNA • What is the mechanism of translational repression by Puf6?

21. Puf6 interfere with 80S assembly • The reporter: mRNA with Renilla luciferase and E3 localization element of ASH1 mRNA • The reporter R-luc-E3 mRNA incubated with Puf6 does not translate in vitro as well as R-luc alone.

22. Puf6 interfere with 80S assembly • Sucrose gradients for in vitro translation assays done with E3 containing mRNA • With H2O get an 80S peak • GMPPNP blocks 60S joining, get increased 48S peak • E3 RNA with Puf6 increases 48S complex and decrease 80S

23. Puf6 interfere with 80S assembly • Competition with cold E3 RNA and Puf6 recovers the 80S peak • In the presence of EDTA 48S complex was not detected with or without Puf6 • Puf6 blocks 80S complex assembly, but not 48S formation. • 60S joining?

24. Fun12p associates with puf6 and is required for ASH1 translation and localization • Puf6 and Fun12 have been found to interact • Puf6 and Fun12 Co-IP together in an RNA dependent manner

25. Fun12p associates with puf6 and is required for ASH1 translation and localization • In fun12 knockout strains: • Ash1p expression in down >80% compared to Pgk1p, and Nop1p • ASH1 mRNA decreased 50% compared to ACT1

26. Fun12p associates with puf6 and is required for ASH1 translation and localization • ASH1 mRNA delocalizes in fun12 mutants • 62% in bud tip • 16% in neck • 12% in mother and bud

27. Both the N-terminal region and PUF domain of puf6p are required for translational repression • C536 (contain PUF domain) • Binds E3 RNA • Binds eIF5B • Represses both R-luc constructs • N120 • Does not contain PUF domain • Need more than the PUF domain

28. Puf6p is phosphorylated by protein kinase CK2 • Puf6p is phosphorylated in yeast cells • lPPAse will remove the phosphates • N120 is phosphorylated when incubated with yeast extracts

29. Puf6p is phosphorylated by protein kinase CK2 • N120 is phosphorylated by casein kinase 2 (CK2) from sea stars • Phosphorylation occurs by ATP or GTP • Phosphorylation sites on N120 match previous predictions for puf6p phosphorylation

30. Puf6p is phosphorylated by protein kinase CK2 • Recombinant Puf6 was phosphorylated in yeast extracts and run on Mass spec • Two site identified Ser34 and Ser35 • These sites • Are in the N120 region • match previous predictions for puf6p phosphorylation • Ser31 could also be a possible phosphorylation site based on previous research

31. CK2 phosphorylation of Puf6p relives translational repression • Puf6 point mutations • Ser to Ala mutation in all three possible phosphorylation sites • Mutant puf6 phosphorylation is down ~90% • Mutant puf6 repressed translation better • Puf6 phosphorylation was decreased by DMAT

32. CK2 phosphorylation of Puf6p relives translational repression • Wild type Puf6 phosphorylation was decreased by DMAT • Translation of R-luc-E3 was reduced in the presence of DMAT

33. CK2 phosphorylation of Puf6p relives translational repression • RNA-binding: • When treated with lppase, Puf6p-TAP purified from yeast extracts retained E3-RNA • Puf6-TAP was not able to maintain mutation E3 RNA • His-Puf6 phosphorylated by CK2 bound E3 less efficiently • Puf6 phosphorylation by CK2 reduces RNA binding, which affects the repressing role.

34. CK2 phosphorylation of Puf6p is required for ASH1 mRNA localization and translation • Localization of Ash1 mRNA: • WT Puf6  bud tip • SApuf6  diffuse • Cka1  diffuse • Cka2 diffuse • CK2 phosphorylation of Pus6 required for localization

35. CK2 phosphorylation of Puf6p is required for ASH1 mRNA localization and translation • CK2 colocalizes in the bud cortex with ASH1 mRNA • CK2 is detectable in the bud cortex before ASH1 mRNA expression began

36. CK2 phosphorylation of Puf6p is required for ASH1 mRNA localization and translation • CK2 moves to the cortex prior to ASH1 mRNA localization • Puf6 is phosphorylated at the bud tip, which allows for localized translation

37. Conclusions • Puf6 binds to the 3’ UTR of ASH1, and blocks translation, by blocking 80S complex formation • Deleting Fun12 decreases ASH1 mRNA expression • The binding of fun12 and mRNA to puf6 is not sufficient for translation repression • CK2 phosphorylation of puf6 is critical for puf6 repression • CK2 catalytic subunits localize to the bud tip prior to ASH1 mRNA localization