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Translational Regulation

Translational Regulation. Sunnie Thompson PTRM 08/26/08. Differences. Translation and Transcription are uncoupled. mRNAs are monocistronic. Ribosomes bind to the RNA at the 5’end (no Shine Dalgarno sequence). mRNA has a 5’cap structure. mRNA has a poly(A) tail (except Histone mRNA)

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Translational Regulation

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  1. Translational Regulation Sunnie Thompson PTRM 08/26/08

  2. Differences Translation and Transcription are uncoupled. mRNAs are monocistronic. Ribosomes bind to the RNA at the 5’end (no Shine Dalgarno sequence). mRNA has a 5’cap structure. mRNA has a poly(A) tail (except Histone mRNA) tRNAmetiis specific for initiation in eukaryotes it is not a formylated tRNAmet Eukaryotic and Prokaryotic Translation Similarities The genetic code is nearly universal, applying to all species on our planet. Protein synthesis begins with an AUG and terminates with UGA, UAA, and UAG.

  3. Differences in Translational Complexity Between Prokaryotes and Eukaryotes

  4. Summary Mechanisms of initiation: Cap-dependent translation Cap-independent translation: Internal ribosome entry sites (IRES) Regulation of initiation: Global eIF2 alpha phosphorylation 4E-BPs Cleavage of eIF4G Message specific IRES Derepression during starvation: GCN4 Steric blockage: IRE/IRP Cap-dependent: CPEB/Maskin Cap-independent: SXL Post-recruitment: hnRNPK/hnRNPE1 Localization: ASH1 miRNA * * * *

  5. mRNA Cap Poly(A) tail Secondary structures IRES uORF Protein/RNA binding sites AUG Kozak consensus sequence: GCC(A/G)CCAUGG Fátima Gebauer & Matthias W. Hentze Nature Reviews Molecular Cell Biology 5, 827-835 (2004)

  6. Scanning model of Cap-dependent Translation Initiation eIF4F: eIF4E – cap binding protein eIF4G – Scaffolding protein eIF4A – Helicase • Ternary complex formation • 43S pre-initiation complex • Recruitment to the 5’end of the mRNA E P A eIF5B Fátima Gebauer & Matthias W. Hentze Nature Reviews Molecular Cell Biology 5, 827-835 (2004) E P A

  7. Closed-loop model * http://departments.oxy.edu/biology/Stillman/bi221/091300/091300_lecture_figures.htm

  8. Scanning model of Cap-dependent Translation Initiation • Ternary complex formation • 43S pre-initiation complex • eIF4F recruits 43S to the 5’end of the mRNA geneerating 48S complex • 43S complex scans down to the AUG • eIF2 positions the met-tRNA in the P-site of the ribosome. eIF1 aids in correct start codon selection • eIF5 hydrolyses eIF2-GTP, release of initiation factors • eIF1A recruits eIF5B-GTP • 60S subunit joins • Hydrolysis of eIF5B-GTP and release of eIF1A and 5B E P A eIF5B Fátima Gebauer & Matthias W. Hentze Nature Reviews Molecular Cell Biology 5, 827-835 (2004) E P A

  9. Regulation of Cap-dependent Translation Initiation X • Reduction of Ternary complex • Inhibition of eIF4E binding to the cap • Cleavage of eIF4G • Inhibition of 43S recruitment • Block 60S subunit joining X X X E P A eIF5B X Fátima Gebauer & Matthias W. Hentze Nature Reviews Molecular Cell Biology 5, 827-835 (2004) E P A

  10. Polysome Analysis { Polysomes { Actin Northern

  11. Global Regulation

  12. Recycling of the ternary complex requires eIF2B Fátima Gebauer & Matthias W. Hentze Nature Reviews Molecular Cell Biology 5, 827-835 (2004)

  13. eIF2 Phosphorylation Reduces Ternary Complex Starvation (GCN2) Viral infection, apoptisis (PKR) ER Stress (PERK) Haemin-regulated inhibitor (HRI) Fátima Gebauer & Matthias W. Hentze Nature Reviews Molecular Cell Biology 5, 827-835 (2004)

  14. GCN4 expression is regulated by uORFs uORFS 1 2 3 4 GCN4 • Encodes a transcriptional activator of genes that regulate AA biosynthesis • Not expressed when AA are abundant • Translation is induced when AA are depleted • The scanning model predicts that the first AUG will be recognized and translated • Mis-sense mutations had no effect so it unlikely that uORFs functioned as a sensor for AA starvation

  15. uORFs function in GCN4 translational control Only need uORF 1 and 4 for regulation

  16. uORFs function in GCN4 translational control Only need uORF 1 and 4 for regulation Removing all uORFs results in constitutive expression of GCN4

  17. uORFs function in GCN4 translational control Only need uORF 1 and 4 for regulation Removing all uORFs results in constitutive expression of GCN4 Removing uORF1 resulted in no expression under starvation conditions

  18. uORFs function in GCN4 translational control Only need uORF 1 and 4 for regulation Removing all uORFs results in constitutive expression of GCN4 Removing uORF1 resulted in no expression under starvation conditions Removing uORF4 results in constitutive expression of GCN4

  19. Abundance of Ternary complex regulates GCN4 Non-starvation Starvation F. Gebauer, M. W. Hentze, Nat Rev Mol Cell Biol5, 827

  20. 4E-BP: eIF4E binding proteins eIF4E binding proteins: 4E-BP Apoptosis Hypoxia * Insulin AA (Leucine) Cell Proliferation F. Gebauer, M. W. Hentze, Nat Rev Mol Cell Biol5, 827 4E-T (eIF4E transporter) inhibits translation and promotes P-body formation

  21. Cleavage of eIF4G SJ Morley, MJ Codwell, MJ Clemens (2006) Cell Death and Diff. 12, 571-584.

  22. Message specific regulation of translation initiation

  23. Viral IRES X&Y=> IRES Trans-acting factors Stoneley and Willis (2004) Oncogene 23, 3200-3207

  24. IRES Mechanisms of Internal Initiation IRES *

  25. Dicistronic Assay for IRES activity

  26. Cap-Independent Translation Initiation • Cellular • Cellular stress: viral infection (PKR), apoptosis, hypoxia • Normal cellular processes: G2/M phase cell cycle • Viral • Genomes: Picornaviruses, Hepatitis C Virus, Cricket paralysis virus • Specific viral messages: HIV, Herpes virus (…. and the list is still growing!)

  27. Cellular IRESs: How do they recruit 40S? • Widely assumed that the Secondary and tertiary Structure allow for interactions with translational machinery (ITAF,eIFs, 40S) • No common Structure • Composed of multiple short modules • Generally 150-300 nts long, exception: 9nt repeated element.

  28. mRNA-rRNA base-pairing Dresios et al. (2006) Nature Struct.& Mol. Bio. 13, 30-34.

  29. IRE/IRP: steric blockage • Binding of the IRP to the IRE sterically hinders the ability of the 43S initiation complex from associating with the mRNA, although eIF4F is bound to the mRNA. • The position of the IRE to the cap is essential for regulation • Another RNA SL and binding partner can functionally replace IRE/IRP F. Gebauer, M. W. Hentze, Nat Rev Mol Cell Biol5, 827

  30. Maskin and Bicoid are mRNA specific 4E-BP (Cap-dependent repression) • CPEB binds Maskin which binds eIF4E and prevents eIF4G binding • Bicoid binds both BRE and eIF4E to repress caudal mRNA F. Gebauer, M. W. Hentze, Nat Rev Mol Cell Biol5, 827

  31. Cap-independent regulation • SXL binds a U-rich sequence in the 5’UTR and 3’UTR • SXL recruits UNR, a co-repressor, that inhibits the association of the 43S complex • 5’UTR bound SXL blocks any scanning by the 43S complex that may have escaped the • SXL-UNR blockage Ann-Bin Shyu. Nature Struct. & Molec. Biol. (2006) 13, 189-190.

  32. Post-recruitment: hnRNPK/hnRNPE1 • Independent of Poly(A) and Cap • Sucrose gradient: 48S complex • Toe-printing: 43S complex at the initiator AUG • hnRNP EI and K prevent 60S joining F. Gebauer, M. W. Hentze, Nat Rev Mol Cell Biol5, 827

  33. Toe-print assay Method used to determine the position of a 43S or 80S complex on an mRNA. F. Gebauer, M. W. Hentze, Nat Rev Mol Cell Biol5, 827 (Oct, 2004). RT A complex is formed (48S or 80S) An oligo is annealed downstream Reverse transcription reaction Analysis of RT stops on a sequencing gel If the AUG is in the P-site of the ribosome expect a stop at 16-18 downstream

  34. Lox mRNA toe-print D. H. Ostareck, A. Ostareck-Lederer, I. N. Shatsky, M. W. Hentze, Cell104, 281

  35. Localization • mRNA must be translationally silent during transport (Puf6p and Khd1p) • mRNA is packaged in “locasomes” for transport along actin bundles • “Zipcodes” lie within the message (typically in the 3’UTR) • Anchoring of the message at the site of localization • Translational activation (phosphorylation of repressors) * N. Paquin, P. Chartrand, Trends Cell Biol18, 105 (Mar, 2008).

  36. Summary Mechanisms of initiation: Cap-dependent translation Cap-independent translation: Internal ribosome entry sites (IRES) Regulation of initiation: Global eIF2 alpha phosphorylation 4E-BPs Cleavage of eIF4G Message specific IRES Derepression during starvation: GCN4 Steric blockage: IRE/IRP Cap-dependent: CPEB/Maskin Cap-independent: SXL Post-recruitment: hnRNPK/hnRNPE1 Localization: ASH1 miRNA

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