Quadruplex structures that result from stacking of guanine quartets in nucleic acids possess such thermodynamic stability that their resolution in vivo is likely to require specific recognition by specialized enzymes. We previously identified the major tetramolecular quadruplex DNA resolving activity in HeLa cell lysates as the gene product of DHX36 (Vaughn, J. P., Creacy, S. D., Routh, E. D., Joyner-Butt, C., Jenkins, G. S., Pauli, S., Nagamine, Y., and Akman, S. A. (2005) J. Biol Chem. 280, 38117-38120), naming the enzyme G4 Resolvase 1 (G4R1). G4R1 is also known as RHAU, an RNA helicase associated with the AU-rich sequence of mRNAs. We now show that G4R1/RHAU binds to and resolves tetramolecular RNA quadruplex as well as tetramolecular DNA quadruplex structures. The apparent K d values of G4R1/RHAU for tetramolecular RNA quadruplex and tetramolecular DNA quadruplex were exceptionally low: 39 ؎ 6 and 77 ؎ 6 pM, respectively, as measured by gel mobility shift assay. In competition studies tetramolecular RNA quadruplex structures inhibited tetramolecular DNA quadruplex structure resolution by G4R1/RHAU more efficiently than tetramolecular DNA quadruplex structures inhibited tetramolecular RNA quadruplex structure resolution. Down-regulation of G4R1/ RHAU in HeLa T-REx cells by doxycycline-inducible short hairpin RNA caused an 8-fold loss of RNA and DNA tetramolecular quadruplex resolution, consistent with G4R1/RHAU representing the major tetramolecular quadruplex helicase activity for both RNA and DNA structures in HeLa cells. This study demonstrates for the first time the RNA quadruplex resolving enzymatic activity associated with G4R1/RHAU and its exceptional binding affinity, suggesting a potential novel role for G4R1/ RHAU in targeting in vivo RNA quadruplex structures.The nucleobase guanine is unique among bases in nucleic acids because of its great tendency toward self-association. Stable cyclic guanine quartets form by self-assembly through Hoogsteen hydrogen bonding between each of the four guanine molecules (N 1 -H and N 2 -H share hydrogen with O 6 and N 7 of the adjacent guanine). In the presence of physiological monovalent cations these quartets can self-associate into vertical stacks that coordinately bond a cation at the center of the G quartets, and the assemblies are further stabilized through stacking interactions (reviewed in Ref. 1). This vertical assembly of G quartets is known as G-quadruplex; this self-associating phenomenon is responsible for the observation that over 30 different derivatives of guanine can form gels in water (2). These are also the forces that stabilize the formation of quadruplex structures in nucleic acids that are also termed G4-DNA and G4-RNA. Since the initial observation by Sen and Gilbert (3) of the formation of tetramolecular G4-DNA, it has been shown that DNA or RNA molecules possessing runs of as few as three guanines in a row can form a variety of extraordinarily stable quadruplex structures (reviewed in Ref. 4). In fact, guanine quadruplex structures create th...

In response to environmental stress, the translation machinery of cells is reprogrammed. The majority of actively translated mRNAs are released from polysomes and driven to specific cytoplasmic foci called stress granules (SGs) where dynamic changes in protein-RNA interaction determine the subsequent fate of mRNAs. Here we show that the DEAH box RNA helicase RHAU is a novel SG-associated protein. Although RHAU protein was originally identified as an AU-rich element-associated protein involved in urokinase-type plasminogen activator mRNA decay, it was not clear whether RHAU could directly interact with RNA. We have demonstrated that RHAU physically interacts with RNA in vitro and in vivo through a newly identified N-terminal RNA-binding domain, which was found to be both essential and sufficient for RHAU localization in SGs. We have also shown that the ATPase activity of RHAU plays a role in the RNA interaction and in the regulation of protein retention in SGs. Thus, our results show that RHAU is the fourth RNA helicase detected in SGs, after rck/p54, DDX3, and eIF4A, and that its association with SGs is dynamic and mediated by an RHAUspecific RNA-binding domain.

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