Probing Structure/Function Relationships in the gurken 5’ UTRPHILLIP FRANKINO, STATE UNIVERSITY OF NEW YORK AT FREDONIA

Review of Protein Synthesis

• Central Dogmao DNA mRNA Protein

Translation• Initiation

Cap Dependent • eIF4E binds to 7m Guanosine CapCap Independent • IRES

IRES Translation

• Internal Ribosomal 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)

Focus of our Studies: 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

spindle-B (spn-B) Mutations Cause Eggshell Ventralization

• Spn-B is essential in DSB repair

• spn-BBU represses grk translation.

• Nutrient Deprivation or Addition of Rapamycin restores grk translation.

TOR Pathway and Rapamycin

TOR

Ra

pa

my

ci

n

4EBP

EIF4E

Cap-Dependent

P

5’ 3’

Rescuing spn-BBU Ventralization

Ferguson et al. 2012

SHAPE Structure of grk

Fountain Lab - Dave DiPalma

Reporter Constructs

grk promoter

grk 5’ UTR

FireflyLuciferase

grk 3’ UTR

Firefly luciferase reporters

Renilla luciferase reportergrk

promoterInR

5’ UTRRenilla

Luciferasegrk

3’ UTR

grk promoter

tub5’ UTR

FireflyLuciferase

grk 3’ UTR

grk promoter

InR5’ UTR

FireflyLuciferase

grk 3’ UTR

Normalization

Tub Native Del SL1 Del SL2 Del Both Mut SL1 Mut SL2 Repair SL2 Mut Both0.0000

0.0500

0.1000

0.1500

0.2000

0.2500

0.3000

0.3500

0.4000

0.4500

Luciferase Assay for Translational Efficiency Ratio FF:Renilla (Yeast) Ratio FF:Renilla (Rapamycin)

Native Del SL1 Del SL2 Del Both Mut SL1 Mut SL2 Repair SL2 Mut Both0.0000

0.0500

0.1000

0.1500

0.2000

0.2500

0.3000

0.3500

0.4000

0.4500

Luciferase Assay for Translational Efficiency Ratio FF:Renilla (Yeast) Ratio FF:Renilla (Rapamycin)

Future Direction

• Polypyrimidine Tract Binding Protein (PTB) Binds Oligopyrimidine Tracts

• PTB Has Stimulatory Effects on IRES Translation (Mitchell et. al., 2005. Spriggs et al., 2009)

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

Acknowledgments

Dr. Fountain

Allison MartinDave DiPalmaCory JacksonDino Salkic

Dr. Ferguson

Jake MerleRamses RodriguezJohn Hasper Chelsea MayJosh BlundonPaige Jacobs

National Science Foundation

Supported by NSF RUI (1243951) to SBF

Fredonia

Questions?