Biochemical Characterization of G4 Quadruplex Telomerase RNA Unwinding by the RNA Helicase RHAU

Booy, Evan P.; McRae, Ewan K.S.; McKenna, Sean Andrew

doi:10.1007/978-1-4939-2214-7_9

Cited by 19 publications

(18 citation statements)

References 36 publications

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“…RHAU 53-105 and full length RHAU were expressed and purified as described previously [ 31 , 43 ]. After removal of the hexahistidine affinity tag by thrombin digestion, the protein was further purified by size exclusion chromatography on a HiLoad Superdex 75 26/60 (ÄKTA GE Healthcare, Mississauga, Canada) in 10 mM HEPES (pH 7.5), 150 mM NaCl (5 mL load volume).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2015

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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.

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Section: Methodsmentioning

confidence: 99%

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

et al. 2015

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“…Another study confirmed that the interaction between DHX36 and the TERC G-quadruplex disrupts the formation of P1 helix, a structure which defines template boundary for reverse transcription. DHX36 was sufficient to unwind the quadruplexes and promote the formation of a stable P1 helix, and DHX36 depletion led to a reduction in average telomere length 138,141 . Besides the function on telomere maintenance, DHX36 regulates translation, although the precise mechanism has not yet been elucidated.…”

Section: Rhau (Dhx36)mentioning

confidence: 99%

RNA G-quadruplexes and their potential regulatory roles in translation

et al. 2016

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DNA guanine (G)-rich 4-stranded helical nucleic acid structures called G-quadruplexes (G4), have been extensively studied during the last decades. However, emerging evidence reveals that 5′- and 3′-untranslated regions (5′- and 3′-UTRs) as well as open reading frames (ORFs) contain putative RNA G-quadruplexes. These stable secondary structures play key roles in telomere homeostasis and RNA metabolism including pre-mRNA splicing, polyadenylation, mRNA targeting and translation. Interestingly, multiple RNA binding proteins such as nucleolin, FMRP, DHX36, and Aven were identified to bind RNA G-quadruplexes. Moreover, accumulating reports suggest that RNA G-quadruplexes regulate translation in cap-dependent and -independent manner. Herein, we discuss potential roles of RNA G-quadruplexes and associated trans-acting factors in the regulation of mRNA translation.

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“…All standard laboratory chemicals and reagents were purchased from Thermo Fisher Scientific. The recombinant full-length RHAU protein, RHAU E335A mutant, RHAU iso.2 , RHAU ⌬RSM , RHAU (1-614), RHAU(1-760), and RHAU(53-105) truncations were cloned using standard molecular biology techniques and expressed and purified as described previously (23,41).…”

Section: Methodsmentioning

confidence: 99%

RNA Helicase Associated with AU-rich Element (RHAU/DHX36) Interacts with the 3′-Tail of the Long Non-coding RNA BC200 (BCYRN1)

Booy¹,

McRae²,

Howard³

et al. 2016

Journal of Biological Chemistry

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RNA helicase associated with AU-rich element (RHAU) is an ATP-dependent RNA helicase that demonstrates high affinity for quadruplex structures in DNA and RNA. To elucidate the significance of these quadruplex-RHAU interactions, we have performed RNA co-immunoprecipitation screens to identify novel RNAs bound to RHAU and characterize their function. In the course of this study, we have identified the non-coding RNA BC200 (BCYRN1) as specifically enriched upon RHAU immunoprecipitation. Although BC200 does not adopt a quadruplex structure and does not bind the quadruplex-interacting motif of RHAU, it has direct affinity for RHAU in vitro. Specifically designed BC200 truncations and RNase footprinting assays demonstrate that RHAU binds to an adenosine-rich region near the 3-end of the RNA. RHAU truncations support binding that is dependent upon a region within the C terminus and is specific to RHAU isoform 1. Tests performed to assess whether BC200 interferes with RHAU helicase activity have demonstrated the ability of BC200 to act as an acceptor of unwound quadruplexes via a cytosine-rich region near the 3-end of the RNA. Furthermore, an interaction between BC200 and the quadruplex-containing telomerase RNA was confirmed by pull-down assays of the endogenous RNAs. This leads to the possibility that RHAU may direct BC200 to bind and exert regulatory functions at quadruplex-containing RNA or DNA sequences.RNA helicase associated with AU-rich element (RHAU), 8 also known as DHX36 and G4R1, is an ATP-dependent DEAHbox RNA helicase initially characterized as a regulator of mRNA stability via binding to the AU-rich element of urokinase plasminogen activator mRNA (1). Subsequent to this, the discovery that RHAU possesses the ability to unwind both DNA and RNA quadruplexes shifted the focus of RHAU to a role in quadruplex biology (2-4).Quadruplexes are stable secondary structures that occur in guanine-rich nucleic acids through the alignment and hydrogen bonding of guanines in multiple tetrad planes (5). A typical requirement for quadruplex formation is the presence of four tracts of three or more guanines that align in parallel or antiparallel strands with short (Ͻ7-nt) interspersed loops (6); however, quadruplexes with longer loops have also been reported (7,8). Quadruplexes have been shown to possess myriad roles in gene transcription and post-transcriptional regulation of both coding and non-coding RNAs (9 -16).The quadruplex binding specificity of RHAU is mediated by a short N-terminal motif, referred to as the RHAU-specific motif (RSM). In addition to the RSM, RHAU contains a catalytic helicase core (DEXDc and HELICc domains) as well as an HA2 domain and oligosaccharide binding (OB) fold. Whereas the RSM confers quadruplex interacting specificity, maximal quadruplex binding affinity requires the presence of the helicase core (17, 18). The DHX36 gene generates two known splice variants of RHAU. Whereas isoform 1 localizes to both nucleus and cytoplasm, isoform 2 lacks 14 amino acids within the helicase domain and is p...

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Biochemical Characterization of G4 Quadruplex Telomerase RNA Unwinding by the RNA Helicase RHAU

Cited by 19 publications

References 36 publications

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

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

RNA G-quadruplexes and their potential regulatory roles in translation

RNA Helicase Associated with AU-rich Element (RHAU/DHX36) Interacts with the 3′-Tail of the Long Non-coding RNA BC200 (BCYRN1)

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