GC-elements controlling HRAS transcription form i -motif structures unfolded by heterogeneous ribonucleoprotein particle A1

HRAS is regulated by two neighbouring quadruplex-forming GC-elements ( hras -1 and hras -2), located upstream of the major transcription start sites (doi: 10.1093/nar/gku 5784). In this study we demonstrate that the C-rich strands of hras -1 and hras -2 fold into i -motif conformations ( i Ms) characterized under crowding conditions (PEG-300, 40% w/v) by semi-transitions at pH 6.3 and 6.7, respectively. Nondenaturing PAGE shows that the HRAS C-rich sequences migrate at both pH 5 and 7 as folded intramolecular structures. Chromatin immunoprecipitation shows that hnRNP A1 is associated under in vivo conditions to the GC-elements, while EMSA proves that hnRNP A1 binds tightly to the i Ms. FRET and CD show that hnRNP A1 unfolds the i M structures upon binding. Furthermore, when hnRNP A1 is knocked out in T24 bladder cancer cells by a specific shRNA, the HRAS transcript level drops to 44 ± 5% of the control, suggesting that hnRNP A1 is necessary for gene activation. The sequestration by decoy oligonucleotides of the proteins (hnRNP A1 and others) binding to the HRAS i Ms causes a significant inhibition of HRAS transcription. All these outcomes suggest that HRAS is regulated by a G-quadruplex/ i -motif switch interacting with proteins that recognize non B-DNA conformations.