Marla Hertz 9-2-08

1. Marla Hertz 9-2-08 Mangus DA. et al, Genome Biology, 2003

2. Closed-loop mRNP • protects the mRNA from degradation • May allow for efficient reinitiating on the same message 5’-3’ exonucleases 3’-5’ exonucleases decapping

3. How is the closed-loop mRNP structure formed and maintained? Factors required for formation: 5’ cap Poly(A) tail eIF4G eIF4E PABP Assume that formation occurs after initiation Mangus DA. et al, Genome Biology, 2003

4. Major claims of the paper • Closed-loop forms during 48S complex formation • Formation requires termination factors but not a termination event • There are two stable closed-loop forms Stage I = 48S complex, Pab1, eIF4G, 4E Stage II = Stage 1 factors plus 60S, eRF1 and 3

5. Closed-loop mRNPs are resistant to cap analogue treatment Closed-loop (resistant) Linear mRNA (sensitive) AAAAA 4G 4G 4G 4G 40S

6. Toeprinting 16-18nt downstream from position of the ribosome 40S cap AUG cyclohexamide RT-PCR product AUG Determines where the ribosome is bound to the message

7. Constructs used in this study Which of these mRNAs form a stable closed loop?

8. Short mRNAs are resistant to cap analogue mRNA length 2135nt 488nt If the mRNA is in a stable closed loop conformation addition of cap analogue, a competitive inhibitor of translation, will not decrease ribosomal recruitment to the message

9. Resistance to cap analogue requires a 5’ cap and a poly(A) tail miniUAA1 Increased resistance with poly(A) length One Pab1 binds to 12A’s

10. Pab1 mutants eIF4G binding eRF3 binding Pab1 wt Pab1-134 Does not bind 4GII Pab1-184 Does not bind 4GI or II Pab1 ΔRRM1 Does not bind poly(A) or 4G Pab1 ΔC-term pab1Δpbp1 Δ no pab1 pbp1Δpab1Δ suppressor = deficient in Poly(A) or 4G binding adapted from: Mangus DA. et al, Genome Biology, 2003

11. Pab1 interactions with 4G and the poly(A) tail are required for cap analogue resistance Reaffirms that 4G and Pab1 are required for complex formation

12. Release factors are required for closed-loop formation Sup35-R419G: eRF3 mutant Sup45-2: eRF1 mutant The mRNA becomes sensitive to cap analogue in yeast extract with mutated eRF1 or 3 Is termination required?

13. Cap analogue resistance occurs independent of termination AAA Sup35-R419G: eRF3 mutant Sup45-2: eRF1 mutant But- formation of the closed loop requires intact eRF1 and 3

14. Cap analogue sensitive mRNA are not translated efficiently Non- polysome polysome Non- polysome polysome Closed loop formation is required to translate effectively

15. There are two closed-loop forms Cyclohexamide GMPPNP

16. Unanswered questions • Is the resistance to cap analogue for only short mRNAs an artifact of the in vitro system? • Do their claims hold up in vivo? • Are eRF1 and 3 bound where they normally bind? • What are they doing? • Does the closed loop structure form before 40S subunit joining?

18. Tumor angiogenesis tumor Vascularized tumor VEGF Growth of vessels towards tumor [O2] blood vessel How is the angiogenic switch activated in tumor cells?

19. Major claims of the paper • Overexpression of 4E-BP and eIF4G in advanced breast cancers • Hypoxia activates the switch from cap-dependent to independent translation in the tumor • Switch to IRES dependent translation advances the cancer

20. 4E-BP globally regulates cap-dependent translation Gebauer and Hentze, Nature Reviews Molecular Cell Biology 5, 2004

21. 4E-BP globally regulates cap-dependent translation Rapamycin Hypoxia Mamane, Y et. al. Oncogene 2006

22. 4E-BP and 4G are overexpressed in advanced tumors Moderately elevated in small tumors Increased level of expression with tumor size Constant expression eIF4A

23. Cell lines used do not completely recapitulate in vivo levels MCF10A- immortalized non-transformed; wt CRL1902- early stage; T1-T2 BT474- late stage; T3 2X fold induction 3X 3X lower, but still rapamycin sensitive

24. Highly transformed cell line has more hypophosphorlyated 4E-BP HyperP HypoP

25. Increased level of VEGF in highly transformed cells • Requires a hypoxic state • Requires 4E-BP and 4G

26. Increased IRES dependent translation during hypoxia EMCV HIF1α

27. 4E-BP expression increases size and vasculature of tumors in BT474 cells

28. Tumor progression slowed in the absence of 4E-BP, eIF4E, or eIF4G

29. Overexpression of 4E-BP and 4G in a mouse tumor model verify in vitro results vascularization 2.5X GFP- VEGF IRES RFP- cap-dependent

30. Unanswered Questions • What is the mechanism behind selective translation of pro-angiogenic IRESs • Subset of ITAFs? • What happens to the specific levels of the mRNA/protein/activity of the inhibitors of angiogenesis • What does this mean for the way we currently treat patients?

31. 4E-BP globally regulates cap-dependent translation mTOR Rapamycin Antiproliferative drug Gebauer and Hentze, Nature Reviews Molecular Cell Biology 5, 2004

32. Many oncogenes and tumor supressors have IRESs Oncogenes L-myc Tumor Supressors p53 Holcik, M., 2004. Current Cancer Drug Targets 4(3):299-311

33. Cells in early stages of transformation send out stress signals Shutdown of cap-dependent translation by overexpression of 4E-BP and 4G IRES-dependent translation increases Shutdown creates environment for selective translation of oncogenes with IRESs Decrease in overall protein synthesis and maintained level of tumor suppressors with IRESs Cancer cell survival Apoptosis/cell repair Requires a secondary mechanism to selective upregulate IRESs that allow for cancer cell survival

34. How is the angiogenic switch activated in tumor cells? Bergers, G and Benjamin, LE, Nat. Rev, 2003

35. What was the biological question addressed? • What major claims do the authors make? • What are the major results that the authors obtained? • Present background and data from paper here • 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?