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

Giulia Miglietta,Susanna Cogoi,Luigi E Xodo,Erik B Pedersen

doi:10.1038/srep18097

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.

Highlights

In the present work we interrogated if the complementary C-rich strands of hras-1 and hras-2 fold into the well known iM conformation[5,6,7,8,9,10,11,12,13,14,15]
In this work we have focused on the complementary C-rich strands hras-1Y and hras-2Y and have investigated if they fold into stable iMs
We have demonstrated that two neighbouring GC-rich elements controlling HRAS expression can form non B-DNA iM structures, which are stable under near-physiological conditions

Summary

Introduction

In the present work we interrogated if the complementary C-rich strands of hras-1 and hras-2 (namely hras1Y and hras-2Y) fold into the well known iM conformation[5,6,7,8,9,10,11,12,13,14,15]. We discovered that the HRAS iMs are recognized by nuclear proteins, including nuclear factor hnRNP A1. This protein, which shows a binding preference for cytosines, unfolds the iM conformation of www.nature.com/scientificreports/. When hnRNP A1 was knocked out in T24 bladder cancer cells by a specific shRNA, the level of HRAS transcript dropped to 44 ± 5% of the control. Our data provide evidence that hnRNP A1, with its unfolding activity against the iM, is an essential factor for the activation of HRAS. The outcome of this work support the notion that HRAS expression is regulated by a G-quadruplex/iM switch that is controlled by proteins

Methods

Results

Conclusion