title: GC-elements controlling HRAS transcription form i-motif structures unfolded by heterogeneous ribonucleoprotein particle A1.
abstract: 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 (iMs) 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 iMs. FRET and CD show that hnRNP A1 unfolds the iM 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 iMs 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.
authors.affiliation: Department of Medical and Biological Sciences, P.le Kolbe 4, 33100 Udine, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Miglietta
authors.affiliation: Department of Medical and Biological Sciences, P.le Kolbe 4, 33100 Udine, Italy.
authors.forename: Susanna
authors.initials: S
authors.lastname: Cogoi
authors.affiliation: Nucleic Acid Center, Institute of Physics and Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark.
authors.forename: Erik B
authors.initials: EB
authors.lastname: Pedersen
authors.affiliation: Department of Medical and Biological Sciences, P.le Kolbe 4, 33100 Udine, Italy.
authors.forename: Luigi E
authors.initials: LE
authors.lastname: Xodo
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D020862:GC Rich Sequence / Q000235:genetics; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D059372:Nucleotide Motifs / Q000235:genetics; D011485:Protein Binding; D058767:Protein Unfolding; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D013816:Thermodynamics; D044366:Transition Temperature
chemical_list: D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)