Investigation of the Regulation of Precursor MicroRNAs through Small Molecule Binding

MicroRNAs (miRNAs) are highly conserved non-coding RNA molecules, approximately 22 nucleotides in length, that are involved in regulating gene expression by binding to target messenger RNAs (mRNAs). The production of mature miRNAs is initiated with the transcription of miRNA genes into primary miRNAs consisting of a stem-loop structure that is then processed to precursor or pre-miRNAs by RNA polymerases. Pre-miRNAs are further cleaved in the cytoplasm by the Dicer enzyme, producing a miRNA-miRNA duplex. The resulting miRNA targets mRNAs by binding to complementary sequences causing the inhibition of mRNA translation or degradation. Recently, it has been shown that pre-miRNAs can form an alternative structure, in addition to the stem-loop structure, that affects processing and leads to underexpression of the miRNA. The alternative structure, known as a G-quadruplex (G4), leads to the inhibition of Dicer-mediated cleavage and the formation of the miRNA. G-quadruplexes are formed by stacking planar G-quartets and are stabilized by Hoogsteen-type hydrogen bonding and pi-pi stacking interactions. Because of the lack of specific recognition elements, this conformation inhibits Dicer activity. In the lab, we are investigating small molecules that bind to the alternative, G-quadruplex structure. Initial high throughput screening of small molecules was done and hit molecules that selectively bind to the G-quadruplex structure of pre-let-7e were identified through that data. Current investigations are aimed at analyzing the binding interaction and effects on enzymatic cleavage from a biophysical and biochemical standpoint using assays such as fluorescence titrations and gel mobility experiments. The pre-let-7e miRNA studied in the lab is part of the let-7 miRNA family first discovered in C. elegans and functions as an important regulator of differentiation, development timing, and tumorigenesis. Additionally, levels of let-7 miRNAs can serve as biomarkers to assist with cancer diagnosis and monitoring. By analyzing the binding of various small molecules to the alternative G-quadruplex structure of pre-let-7e, we can explore the role of these non-canonical structures in the regulation of miRNA processing and can investigate the therapeutic potential of these molecules in cancers with misregulated let-7e.