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.
Highlights
G-quadruplexes (G4) are four-stranded structures of DNA or RNA in which one guanine base from each chain associates via cyclic Hoogsteen [1] hydrogen bonding to form planar quartets
Q2RNA elutes as a compact dominant peak with a shoulder corresponding to larger hydrodynamic volumes (Fig 1) from the HiLoad Superdex 75 26/60 column
To understand the potential differences between RNA and DNA G4 recognition, we investigated the DNA equivalent to Q2RNA (Q2DNA)
Summary
G-quadruplexes (G4) are four-stranded structures of DNA or RNA in which one guanine base from each chain associates via cyclic Hoogsteen [1] hydrogen bonding to form planar quartets. Genome-wide computational analysis has identified more than 300,000 potential intramolecular G4-forming sequences in the human genome [9, 13] and revealed a higher prevalence of these sequences in functional genomic regions such as telomeres, promoters [10, 14], untranslated regions (UTRs) [15, 16] and introns [17] Taken together, these observations suggest that G4 structures participate in regulating myriad biological processes
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