Biophysical Characterization of G-Quadruplex Recognition in the PITX1 mRNA by the Specificity Domain of the Helicase RHAU

PLoS One. 2015 Dec 9;10(12):e0144510. doi: 10.1371/journal.pone.0144510. eCollection 2015.

Abstract

Nucleic acids rich in guanine are able to fold into unique structures known as G-quadruplexes. G-quadruplexes consist of four tracts of guanylates arranged in parallel or antiparallel strands that are aligned in stacked G-quartet planes. The structure is further stabilized by Hoogsteen hydrogen bonds and monovalent cations centered between the planes. RHAU (RNA helicase associated with AU-rich element) is a member of the ATP-dependent DExH/D family of RNA helicases and can bind and resolve G-quadruplexes. RHAU contains a core helicase domain with an N-terminal extension that enables recognition and full binding affinity to RNA and DNA G-quadruplexes. PITX1, a member of the bicoid class of homeobox proteins, is a transcriptional activator active during development of vertebrates, chiefly in the anterior pituitary gland and several other organs. We have previously demonstrated that RHAU regulates PITX1 levels through interaction with G-quadruplexes at the 3'-end of the PITX1 mRNA. To understand the structural basis of G-quadruplex recognition by RHAU, we characterize a purified minimal PITX1 G-quadruplex using a variety of biophysical techniques including electrophoretic mobility shift assays, UV-VIS spectroscopy, circular dichroism, dynamic light scattering, small angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our biophysical analysis provides evidence that the RNA G-quadruplex, but not its DNA counterpart, can adopt a parallel orientation, and that only the RNA can interact with N-terminal domain of RHAU via the tetrad face of the G-quadruplex. This work extends our insight into how the N-terminal region of RHAU recognizes parallel G-quadruplexes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • AU Rich Elements
  • Biophysics
  • Circular Dichroism
  • DEAD-box RNA Helicases / physiology*
  • DNA / metabolism
  • G-Quadruplexes*
  • Humans
  • Paired Box Transcription Factors / metabolism
  • Protein Binding
  • RNA, Messenger / analysis*

Substances

  • Paired Box Transcription Factors
  • RNA, Messenger
  • homeobox protein PITX1
  • DNA
  • DHX36 protein, human
  • DEAD-box RNA Helicases

Grants and funding

Funded by the Cancer Research Society (Canada) #18206. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.