Analyzing the interaction between RNA and RBPs (RNA-binding proteins) by NMR spectroscopy: the introduction of small molecules as third wheel

Hu (or ELAV-L) proteins belong to the class of RNA- Binding Proteins (RBPs) whose main role is to form complexes with mRNAs thus modulating the production of specific proteins.1 HuR is a member of this family, and its target mRNAs encode proteins involved in cell growth, tumorigenesis, angiogenesis, tumor inflammation, invasion and metastasis. HuR overexpression is correlated with high-grade malignancy and poor prognosis in many tumor types.2 Previous studies3 led to the identification of small, drug-like molecules able to interfere with HuR - RNA complexes by applying different approaches (in silico studies combined with STD NMR, virtual screening, and fragment-based drug discovery). We present here the interaction studies between members of the new series of designed and synthetized ligands with HuR obtained by Saturation Transfer Difference (STD) NMR. Moreover, we report on NMR competition studies in presence of RNA fragment, to confirm that the ligands can bind HuR in the RNA binding site. To overcome the limitations due to RNA handling, that can affect the NMR results, we exploited the use of Peptide Nucleic Acid (PNA) fragments as RNA mimics, using the RNA sequence of TNFα transcript (PDB code: 4ED5). Results clearly show that in STD experiments PNA can be used instead of RNA. The method herein proposed is a worth alternative for identifying molecules that can effectively modulate HuR-RNA complexes, as potential pharmacological agents. Furthermore, this approach will support research on more RNA-binding proteins.