Talk abstracts

Talk on Saturday 09:45-10:00am submitted by Debmalya Bhattacharyya

Modulation of gene expression by inducing G-quadruplex structures in the 5'-UTR

Debmalya Bhattacharyya ( Chemistry and Biochemistry, Kent State University), Kim Nguyen (University of Kentucky), Soumitra Basu ( Chemistry and Biochemistry, Kent State University)

Abstract:
Eukaryotic initiation factor-4E (eIF-4E) is a cap binding protein and is a component of the eIF-4F complex that plays an important role in translation initiation. eIF-4E is overexpressed in multiple cancer types including prostrate, breast, stomach, colon and lungs.1 Knock down of eIF-4E suppresses tumor growth, enhances apoptosis and increases sensitivity to chemotherapeutics in human breast cancer cells2 which makes it a potent therapeutic target. The presence of a G-quadruplex (GQ) in the 5′-untranslated region (UTR) of an mRNA mostly represses translation.3 Intermolecular GQ structures can be induced in an mRNA to repress the translation. The selective induction of GQ in an mRNA sequence can be achieved by an attached guiding sequence. The above strategy opens up an entirely new domain of cancer therapeutics. We show here that hybrid (DNA:RNA) intermolecular G-quadruplex structure can be induced by targeting a section of the 5prime-UTR of the eIF-4E mRNA with a DNA. The formation of the intermolecular G-quadruplex was established by circular dichroism (CD), gel shift, chemical and enzymatic footprinting. Thermodynamic parameters calculated from CD melting shows deltaG to be -19.22±2.47 kJ which indicates a stable structure under physiological conditions. The specificity of the targeting was established by downregulation of the expression of the reporter gene in a dual luciferase reporter. We also show the inhibition of eIF-4E dependent translation compared to an IRES dependent translation in a bicistronic reporter plasmid. Treatment with the GQ inducing sequence showed significant decrease in cell viability in cancer cells. Our data indicate that expression of specific genes can modulated by inducing G-quadruplex structures in cancer cells.

References:
1. Hsieh, A. C. & Ruggero, D. Targeting Eukaryotic Translation Initiation Factor 4E (eIF4E) in Cancer. Clin. Cancer Res.16, 4914-4920 (2010).
2. Dong, K. et al. Tumor-specific RNAi targeting eIF4E suppresses tumor growth, induces apoptosis and enhances cisplatin cytotoxicity in human breast carcinoma cells. Breast Cancer Res. Treat.113, 443-456 (2009).
3. Bugaut, A. & Balasubramanian, S. 5prime-UTR RNA G-quadruplexes: translation regulation and targeting. Nucleic Acids Res40, 4727-4741 (2012).

Keywords: Translation repression, eIF-4E, G-quadruplex strucutres, Cancer,