Using NMR to Identify Structural Features of Lin28-Regulated miRNAs and mRNAs and as a Tool for Comparing Differences in Cellular Metabolism

Abstract

Part 1 of this thesis seeks to identify shared structural features of Lin28-regulated miRNAs and mRNAs. Lin28 is an evolutionarily conserved, RNA binding protein, highly expressed in stem cells and poorly differentiated cancers, that inhibits differentiation and helps maintain stem cell properties. Lin28 binds to both the loops of let-7 precursors to block let-7 biogenesis and to Lin28 responsive elements (LREs) in mRNAs either to enhance or inhibit translation. Lin28 RNA binding properties are not well defined. We used NMR spectroscopy, fluorescence assays and bioinformatics to identify common features of Lin28 targets. We show that Lin28 binds G-rich sequences that have properties of G-quartets (G4s). Based on mutational analysis, we show that G4s are important for Lin28 binding. Upon binding, Lin28 may unwind the G4 structure. Our findings suggest that Lin28 recognizes G-quartets in the RNAs it regulates and might function to unwind them. In part 2 of this thesis we use an unbiased NMR metabolite screening method to identify glucose metabolites differentially produced in BPLER and HMLER isogenic triple negative breast cancer cell lines that have dramatic differences in tumor initiating capacity. N-acetylneuraminic acid (Neu5Ac), a sugar added to the end of glycosylation chains, is much more abundant in BPLER than HMLER cells. Manipulating Neu5Ac expression using neuraminidase or siRNA knockdown of the Neu5Ac biosynthetic enzymes N-acetylneuraminic acid synthase (NANS) or cytidine monophosphate N-acetylneuraminic acid synthase (CMAS), reduces in vitro invasivity of BPLER cells. CMAS protein is also increased in BPLER relative to HMLER. Overexpressing CMAS in HMLER cells increases their invasiveness. Moreover, stable knockdown of CMAS blocks BPLER tumor growth in xenografted mice. Thus increased Neu5Ac synthesis is linked to tumor initiation and invasivity in a human triple negative breast cancer cell line.