Recognition and Resolution of KRAS 5'UTR RNA G-Quadruplexes by hnRNPA1

Abstract

The KRAS oncogene, central to cellular signaling via MAPK and PI3K-AKT pathways, is a notorious cancer driver frequently activated in pancreatic, colorectal, and lung carcinomas. Regulation of human KRAS oncogene expression is important due to its capital role in cell growth, proliferation, and survival. Misregulation of its expression contributes directly to the development and progression of multiple types of cancer. In previous studies, the role of G-quadruplexes elements in both the promoter and 5 UTR regions have shown to play important roles in KRAS expression, particularly when these G4s elements interact with regulatory protein hnRNPA1. In this study, we reveal that KRAS expression is also modulated at the post-transcriptional level through the formation of RNA G-quadruplexes (rG4s) situated at the 5 untranslated region (5UTR) of the mRNA. Biophysical and binding studies were carried out to probe the interaction. Through isothermal titration calorimetry (ITC), we quantified a strong binding affinity between the UP1 domain of hnRNPA1 and short-nucleotide RNA segments capable of adopting different G-quadruplex fold. The binding interaction is characterized by a favorable Gibbs free energy change in the range of {Delta}G {approx} -32 to -34 kJ/mol, suggesting a specific and energetically favorable association. One-dimensional and two-dimensional 1H-15N HSQC NMR spectroscopy revealed pronounced chemical shift changes in residues of both RNA recognition motifs (RRMs) of UP1, signifying direct contact with the rG4 structure.