Title

G Quadruplex RNA Structures in PSD-95 Mrna: Potential Regulators of Mir-125a Seed Binding Site Accessibility

Defense Date

12-15-2014

Graduation Date

Spring 1-1-2015

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

School

Bayer School of Natural and Environmental Sciences

Committee Chair

Rita MIhailescu

Committee Member

Ellen Gawalt

Committee Member

Michael Cascio

Committee Member

Gary Bassell

Keywords

Pure sciences, Fmrp, Fragile x syndrome, Mir-125a, Psd-95

Abstract

Fragile X syndrome (FXS) is the most common inherited form of intellectual disability caused by the CGG trinucleotide expansion in the 5'-untranslated region of the FMR1 gene on the X chromosome, that silences the expression of the Fragile X mental retardation protein (FMRP). FMRP has been shown to bind to a G rich region within the PSD-95 mRNA which encodes for the postsynaptic density protein 95 (PSD-95), and together with the microRNA miR-125a, to play an important role in the reversible inhibition of the PSD-95 mRNA translation in neurons. The loss of FMRP in Fmr1 KO mice disables this translation control in the production of the PSD-95 protein. Interestingly, the miR-125a binding site on PSD-95 mRNA is embedded in the G rich region bound by FMRP and postulated to adopt one or more G quadruplex structures. In this study we have used different biophysical techniques to validate and characterize the formation of parallel G quadruplex structures and binding of miR-125a to its complementary sequence located within the 3'-UTR of PSD-95 mRNA. Our results indicate that the PSD-95 mRNA G rich region folds into alternate G quadruplex conformations. MiR-125a forms a stable complex with PSD-95 mRNA, as evident by characteristic Watson-Crick base pairing that co-exists with one of the G-quadruplex forms, suggesting a novel mechanism for G-quadruplex structures to regulate the access of miR-125a to its binding site. The FMRP regulation of PSD-95 mRNA translation is complex, being mediated by the FMRP phosphorylation. FMRP in its phosphorylated state and miR-125a containing RISC synergistically inhibit the PSD-95 mRNA translation, whereas upon FMRP dephosphorylation triggered by synaptic input, the RISC complex is no longer bound to PSD-95 mRNA, allowing for its translation to occur. While the requirement for FMRP in the regulation of PSD-95 mRNA translation is clearly established, the exact mechanism by which this is achieved is not known. We have shown that an unphosphorylated FMRP and its phosphorylated mimic bind to the proposed G quadruplexes with high affinity, without disrupting the binding of miR-125a to its complementary sequence within PSD-95 mRNA. We propose a novel mechanism by which the translation of PSD-95 mRNA is controlled by the interactions between the AGO2 protein from the RISC complex and the phosphorylated FMRP that brings the miR-125a in the close proximity to its complementary sequence located between the two stable G quadruplexes.

Format

PDF

Language

English

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