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Frankino SURC Presentation
This document discusses experiments probing the structure and function of the 5' untranslated region (UTR) of the gurken mRNA in Drosophila oogenesis. It finds that the gurken 5' UTR contains an internal ribosomal entry site (IRES) that allows for cap-independent translation, similarly to other essential eukaryotic mRNAs. Using luciferase reporter assays and SHAPE structure mapping, the study identifies two stem-loop structures in the gurken 5' UTR that are important for its translational efficiency, particularly under nutrient stress. Future work aims to investigate whether the polypyrimidine tract binding protein homolog Hephaestus regulates gurken translation.
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Frankino SURC Presentation
- 1. Probing Structure/Function Relationshipsin the gurken 5’ UTR PHILLIP FRANKINO, STATE UNIVERSITY OF NEW YORK AT FREDONIA
- 2. Review of ProteinSynthesis • Central Dogma o DNA mRNA Protein Translation • Initiation Cap Dependent • eIF4E binds to 7m Guanosine Cap Cap Independent • IRES
- 3. IRES Translation • InternalRibosomal Entry Site • This is a cap-independent mechanism of translation initiation • Examples Studied in Essential Eukaryotic mRNAs • Cell Stress • Nutrient Deprivation • Drosophila InR (Marr et al. 2007) • Human InR (Spriggs et al. 2009) • Human Hsp70 (Rubtsova et al. 2003) • Human VEGF (Stein et al. 1998)
- 4. Focus of ourStudies: Gurken • Gurken (Grk) is an EGFR ligand essential in Drosophila Oogenesis • Without Grk – Absence of Dorsal Appendages Dark Field: Rodriguez and Ferguson Gurken Ferguson et al. 2012Ferguson et al. 2012
- 5. spindle-B (spn-B) MutationsCause Eggshell Ventralization • Spn-B is essential in DSB repair • spn-BBU represses grk translation. • Nutrient Deprivation or Addition of Rapamycin restores grk translation.
- 6. TOR Pathway andRapamycin TOR 4EBP EIF4E Cap-Dependent P 5’ 3’
- 7. Rescuing spn-BBU Ventralization Fergusonet al. 2012
- 8. SHAPE Structure ofgrk Fountain Lab - Dave DiPalma
- 9. Reporter Constructs grk promoter grk 5’ UTR Firefly Luciferase grk 3’UTR Firefly luciferase reporters Renilla luciferase reporter grk promoter InR 5’ UTR Renilla Luciferase grk 3’ UTR grk promoter tub 5’ UTR Firefly Luciferase grk 3’ UTR grk promoter InR 5’ UTR Firefly Luciferase grk 3’ UTR Normalization
- 10. 0.0000 0.0500 0.1000 0.1500 0.2000 0.2500 0.3000 0.3500 0.4000 0.4500 0.5000 Tub Native DelSL1 Del SL2 Del Both Mut SL1 Mut SL2 Repair SL2 Mut Both Luciferase Assay for Translational Efficiency Ratio FF:Renilla (Yeast) Ratio FF:Renilla (Rapamycin) 0.0000 0.0500 0.1000 0.1500 0.2000 0.2500 0.3000 0.3500 0.4000 0.4500 0.5000 Native Del SL1 Del SL2 Del Both Mut SL1 Mut SL2 Repair SL2 Mut Both Luciferase Assay for Translational Efficiency Ratio FF:Renilla (Yeast) Ratio FF:Renilla (Rapamycin)
- 11. Future Direction • PolypyrimidineTract Binding Protein (PTB) Binds Oligopyrimidine Tracts • PTB Has Stimulatory Effects on IRES Translation (Mitchell et. al., 2005. Spriggs et al., 2009)
- 12. Future Direction • Hephaestus(Heph)– PTB Homolog • Required for Grk localization, but not grk mRNA localization (McDermott and Davis 2013) • Cross heph Mutants to spn-B mutants • Test for Capacity to Suppress Ventralized Eggshell Morphology • Localization of grk
- 13. Acknowledgments Dr. Fountain Allison Martin DaveDiPalma Cory Jackson Dino Salkic Dr. Ferguson Jake Merle Ramses Rodriguez John Hasper Chelsea May Josh Blundon Paige Jacobs National Science Foundation Supported by NSF RUI (1243951) to SBF Fredonia
- 14.
Editor's Notes
- #4 Cap dependent translation requires a large amount of energy, with a need for a handful of translation factors.
- #6 Spn-B represses cap dep
- #9 So what are we doing to characterize this suspected IRES? This is a preliminary SHAPE structure of Gurken. SHAPE stands for Selective 2’ Hydroxyl Acylation and Primer Extension – It is a way to predict secondary structure that may form in the mRNA, under physiological conditions.