{'fraction_non_alphanumeric': 0.036100453435647016, 'fraction_numerical': 0.03117370073247297, 'mean_word_length': 4.984085572658492, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Biophysical Characterization of G-Quadruplex Recognition in the PITX1 mRNA by the Specificity Domain of the Helicase RHAU

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.


Introduction
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. Two or more such quartets hydrophobically stack on top of each other to form the G4 and are stabilized by the presence of a mandatory monovalent cation (typically K + ) in the center between the planes [2]. G4s in DNA and RNA can adopt a parallel, anti-parallel, or hybrid (mixture of both parallel and antiparallel) strand orientation [3]. Biophysical studies and highresolution structures of RNA G4s reveal that they are thermodynamically more stable in vitro than their DNA counterparts under near-physiological conditions because of the 2 0 -OH on the ribose sugar that permits additional hydrogen bonds to form. As a result, RNA G4s preferentially adopt a parallel conformation over an antiparallel one [4][5][6].
A survey of the evolutionary conservation of DNA and RNA motifs revealed that G4 motifs are significantly conserved in the genomes of living organisms [7][8][9][10], and it was recently demonstrated that G4 formation is regulated dynamically during cell-cycle progression [11,12]. Accumulating evidence suggests an important role of G4 structures in regulating gene expression [10]. 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.
To expand our understanding of biologically relevant RNA G4 recognition by the helicase RHAU, we previously performed an RNA co-immunoprecipitation screen and identified the messenger RNA (mRNA) for the protein Pituitary homeobox 1 (PITX1, P-OTX, backfoot) [26]. PITX1 functions as a transcription factor that plays a pivotal role in the differentiation of the developing pituitary gland, craniofacial structures and hind limbs in early embryonal development [34][35][36][37]. Recently, malformations in the lower limbs could be attributed to mutations in the PITX gene [38]. Deletions in PITX1 cause a spectrum of lower-limb malformations including mirror image polydactyly. PITX1 expression is down regulated in a number of tumor types including lung, colorectal, gastric and esophageal cancer and reduced PITX1 expression has been correlated with decreased overall patient survival [39][40][41]. Most interestingly, the PITX1 mRNA possesses three distinct G4 forming sequences in the 3 0 -untranslated region (UTR) of its mRNA (Q1: PITX1 1371-1400 , Q2: PITX1  , and Q3: PITX1 2044-2079 ). These G4s play roles in the recruitment of RHAU to the PITX1 mRNA and ultimately regulate PITX1 protein translation [26]. In cell lysates and with purified components, both RHAU and RHAU  can interact with Q1, Q2, or Q3. Here, we characterize the Q2RNA/RHAU 53-105 complex using a combination of electrophoretic mobility shift assays, UV-VIS spectroscopy, circular dichroism, dynamic light scattering, small angle X-ray scattering (SAXS) and nuclear magnetic resonance spectroscopy. Our integrated approach suggests that the RSM recognizes the planar guanine quartet face of parallel RNA G4s.

Protein expression and purification
RHAU  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). Isotopically enriched 15 N-labelled RHAU  was overexpressed in M9 minimal medium according to the method described previously [31]. The extinction coefficient (7020 M -1 cm -1 ) was used to determine the protein concentration by measuring absorbance at 280 nm, and confirmed by Bradford assay.

G4-protein complex preparation
G4s and RHAU  were diluted to 10 μM in the corresponding buffers described above, mixed in an equimolar ratio, and agitated slowly on a rotator for 15 min at room temperature. Complexes were separated from individual components by SEC on a HiLoad Superdex 75 26/ 60 column (GE-Healthcare, Mississauga, Canada) for purification in 10 mM Tris (pH 7.5), 100 mM KCl, and 1 mM EDTA. Both protein and nucleic acid components in the complex were confirmed by gel electrophoresis following complex purification. The concentration of the complex was determined by UV absorption using the extinction coefficient ε 260nm of Q2 RNA, since the nucleic acid dominates the spectrum.

Microscale thermophoresis (MST)
Binding reactions were prepared in 50mM Tris-HCl buffer (pH 7.8) with 150 mM NaCl, 10 mM MgCl 2 , 0.5% Glycerol and 0.05% Tween to a total volume of 20 μL. RHAU 53-105 was diluted 16 times by 2:1 serial dilution to achieve concentrations ranging from 250-0.6nM, and mixed with fluorescent 3'-FAM labeled Q2RNA (purchased from Integrated DNA Technologies, Coralville, Iowa) was held constant at 25 nM. Premium coated capillaries (NanoTemper Technologies, San Francisco, CA) were used for all measurements. Measurements were performed at an LED power 90% and MST-IR power 40% on the Monolith NT.115 instrument under room temperature conditions (21.5°C). For each run the infrared laser was applied for 35 seconds and the reverse T-Jump data signals of the MST-traces were fitted using the law of mass action for 1:1 binding to obtain K D values.

Dynamic Light Scattering (DLS)
DLS data were collected on a Nano-S Dynamic Light Scattering system (Malvern Instruments Ltd., Malvern, UK) as previously reported [44]. Samples were filtered through a 0.1-μm filter (Millipore) and equilibrated for 5 minutes at 20°C before measurements. 15 measurements were made per sample, and for each condition three independent samples were tested.
Thermal difference spectra UV/VIS spectra were obtained on a dual beam Evolution 260 Bio UV-Visible spectrophotometer (Thermo Scientific). Q2RNA and Q2DNA (2 μM) in 10 mM Tris (pH 7.5), 100 mM KCl, and 1 mM EDTA were measured in triplicate and background corrected against spectra of buffer alone. Thermal difference spectra (TDS) were generated by subtracting buffer-corrected spectra at 20°C from those at 90°C. For direct comparison between Q2RNA and Q2DNA, differences were normalized to the maximum observed absorbance value, as previously suggested by Mergny et. al. [45].

Circular dichroism spectropolarimetry (CD)
All spectra were recorded on a calibrated Alfa Aesar J-810 spectropolarimeter (Jasco Inc., USA) from 200-340 nm in a 1.0 mm cell and a 32 s integration time. Sample concentrations were kept at 20 μM in 10 mM Tris (pH 7.5), 100 mM KCl, 1 mM EDTA. Measurements were performed in triplicate and baseline-corrected by subtraction of the buffer alone. Circular dichroism thermal melting curves were generated in the same buffer, following the ellipticity at 262 nm with spectra normalized by the number of nucleotides (glycosidic bonds) per unit volume.
Small angle X-ray scattering (SAXS) SAXS data were collected using a Rigaku 3-pinhole camera (S-MAX3000) equipped with a Rigaku MicroMax + 002 microfocus sealed tube (Cu-Kα radiation at 1.54 Å) and Confocal Max-Flux (CMF) optics operating at 40 W as previously reported [46]. Scattering data were collected at the following sample concentrations; Q2RNA and Q2DNA (0.8, 1.1, and 1.7 mg/ ml), and Q2RNA/RHAU 53-105 complex (1.3 and 1.5 mg/mL) in 10 mM Tris (pH 7.5), 100 mM KCl, 1 mM EDTA. The raw intensity data were integrated with the SAXSGUI software package (JJ X-Ray Systems A/S, LyngBy, Denmark). Buffer subtraction and merging of data of multiple concentrations were performed using the program PRIMUS [47]. The pair distance distribution function plot, root mean square radius of gyration (r G ) and the maximum particle dimension (D max ) were obtained using the program GNOM [48]. Ab initio shape modeling was performed using the program DAMMIF based on a simulated annealing protocol [49,50]. Twenty models for each entity were then generated, rotated, aligned and averaged using the program DAMAVER [51]. HYDROPRO [52] was used to calculate solution hydrodynamic properties of the averaged-filtered models using a similar approach as outlined previously [46]. Sample quality was confirmed for each sample before and after data collection by gel electrophoresis and DLS.

Q2RNA and Q2DNA each adopt a single, monomeric conformation
Synthetic Q2RNA was heat denatured, cooled, and purified by size exclusion chromatography (see Materials and Methods). 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. The nucleic acid sequences used in this study are presented in (Fig 2A). Native gel electrophoresis confirmed that the dominant peak contains a single RNA conformation ( Fig  2B). To understand the potential differences between RNA and DNA G4 recognition, we also investigated the DNA equivalent to Q2RNA (Q2DNA). Using an identical procedure, Q2DNA eluted in a single symmetric peak that contains a single conformation as determined by native gel electrophoresis (Figs 1 and 2B).
Q2RNA, but not Q2DNA, stains with a dye specific for parallel G4 To determine whether the purified nucleic acids adopt a parallel G4 conformation, we employed native gel electrophoresis in combination with N-methyl mesoporphyrin IX (NMM), a dye specific to parallel G4 conformations. The crystal structure of NMM bound to a parallel DNA G4 demonstrates the selectivity for parallel G4s [53]. To confirm the validity of the approach, non-G4 double-stranded RNA (dsRNA) and a known parallel RNA G4 (hTR 1- Q2RNA adopts a parallel, while Q2DNA adopts an alternate, G4 conformation To confirm G4 formation by Q2RNA, thermal difference spectra (TDS) were generated by subtracting the UV absorption spectrum of the folded state (recorded at 20°C) from the spectrum of the partially denatured state (measured at 90°C). Specific nucleic acid conformations result in specific TDS, generally reflecting the conformational change of the molecule in solution due to a disruption in base-stacking interactions. The TDS obtained for Q2RNA demonstrated features characteristic of G4s [45] with a minimum at 297 nm and two maxima at 240 and 276 nm (Fig 3). Interestingly, TDS analysis of Q2DNA showed similar overall features suggestive that it also adopts a G4 structure.
Next, we performed circular dichroism (CD) spectroscopy on the partially denatured and native states at 80°C and 20°C, respectively, in the same buffer as used for TDS analysis. The far-UV CD spectrum of Q2RNA at 20°C presented features consistent with previously characterized parallel G4s, with an ellipticity minimum at 242 nm and maximum at 264 nm [54] (Fig  4A). At 80°C similar overall spectral features were observed, however the intensity was modestly muted (approximately 45% at 264 nm), presumably due to partial unstacking of the G4 structure. Q2DNA has similar overall features to Q2RNA at 20°C with the prominent exception of an additional maxima at 290 nm (Fig 4A). The Q2DNA spectrum is consistent with the  features of a group II G4 spectrum that has three parallel and one antiparallel strands [55][56][57]. At 80°C, the Q2DNA is almost completely denatured. To determine the relative stabilities of the RNA and DNA G4s, CD spectra were collected during the process of thermal melting ( Fig  4B). Q2RNA was significantly more resistant to denaturation than its DNA counterpart, but the melting profile is similar to that of previously characterized RNA G4s [31]. We conclude that Q2RNA adopts, as expected, a parallel G4 conformation, whereas Q2DNA assumes a hybrid-type G4 structure with parallel and antiparallel strands.

RHAU interacts with Q2RNA but not its DNA counterpart
Previously, an N-terminal truncation of RHAU (RHAU 53-105 ) containing the RSM has been identified to play a significant role in the recognition of G4s [31,33]. To confirm the original observation, we performed electrophoretic mobility shift assays (EMSA) between Q2RNA and either RHAU  or full-length RHAU (Fig 5A). Both RHAU  and full-length RHAU shift Q2RNA towards a higher molecular weight species in a concentration dependent manner. We observed a higher affinity with full-length RHAU than with the truncated version (as expected). Interestingly, the DNA counterpart, Q2DNA, did not show any appreciable affinity for RHAU 53-105. (Fig 5A). Microscale thermophoresis measurements were used to determine a dissociation constant of 1.7±0.3 nM for the RHAU 53-105 complex with fluorescently labeled 3'-FAM-Q2RNA (Fig 5B).
To further characterize nucleic acid-protein complexes, we prepared pure RHAU 53-105 as well as its complex with Q2RNA or Q2DNA, and subjected them to size exclusion chromatography (Fig 1). RHAU  and its complex with Q2RNA eluted as single peaks, with an expected increase in hydrodynamic size accompanying complex formation. Not surprisingly, no complex formation was observed between Q2DNA and RHAU 53-105 (data not shown). The association of N-terminal RHAU with Q2RNA does not disrupt G4 structure To determine whether RHAU  binding disrupts G4 structure we performed a CD experiment on the purified Q2RNA/RHAU 53-105 complex (Fig 6). No significant differences were observed between CD spectra from Q2RNA/RHAU  and Q2RNA in the region unique to nucleic acids (~250-320 nm), suggesting that the G4 remains intact upon protein binding.
Solution structures of G4s and their complexes with RHAU  To further understand the recognition of G4s by RHAU 53-105 , we used SAXS to study Q2RNA, Q2DNA, and the Q2RNA/RHAU 53-105 complex purified by size exclusion chromatography. DLS was employed as an initial quality control step to ensure sample monodispersity over the range of concentrations used for SAXS acquisition (Fig 7A). Decreasing hydrodynamic radii (r H ) were observed for the molecules in the following order: Q2RNA/RHAU 53-105 complex (3.65 nm), Q2RNA (2.01 nm) and Q2DNA (1.65 nm) ( Table 1). Samples did not display any significant self-association in the concentration range subsequently used for SAXS analysis, suggesting suitability for further structural studies ( Fig 7B).
SAXS data for Q2RNA, Q2DNA AND Q2RNA/RHAU 53-105 complex collected at multiple concentrations were merged to obtain a single scattering profile (Fig 7C). The pair distance distribution function, P(r), which represents a histogram of all observed distances between electron pairs in the molecule was obtained from merged data using program GNOM (Fig 7D). Both Q2RNA and Q2DNA demonstrate a P(r) plot consistent with a globular structure, whereas the Q2RNA/RHAU 53-105 complex likely adopts an extended conformation based on the elongated tail at longer distances. From this analysis, the radius of gyration (r G ) and maximum particle dimension (D max ) were determined (Table 1), and used as constraints to generate 20 individual low-resolution models. Individual models, with the chi (χ) values shown in Table 1, were rotated and superimposed to obtain an averaged solution conformation (Fig 8). Excellent superimposition of individually calculated models were confirmed by the normalized spatial discrepancy (NSD) parameter ( 0.63) for each molecule ensemble. Both RNA and DNA G4s adopt disc-shaped structures with concave bevels at the top and bottom, while the Q2RNA/RHAU 53-105 complex adopts an extended shape. Superposition of the previously determined RHAU 53-105 solution structure by SAXS onto the Q2RNA/RHAU 53-105 complex suggests that G4 recognition is occurring via one of the termini of the protein.

Discussion
G4s were predicted to play key roles in a number of biological activities including the regulation of gene transcription and translation [10], and evidence for that has accumulated in recent years both in vitro and in vivo [15,58,59]. Various proteins interact specifically with G4s, suggesting they fulfill important functions in cellular processes [60]. RHAU has been observed in a number of contexts to interact with RNA G4s, but the mechanism of how it recognizes and unwinds these structures is not well characterized. We have chosen as our model system a specific G4 (Q2RNA) found in the 3'-UTR of the PITX1 mRNA, primarily because its interaction with RHAU in a cellular context is established [26]. Based on several methods, including staining with an orientation-specific dye, TDS, and spectropolarimetry, Q2RNA is a parallel G4, and adopts a compact, disc-shaped conformation in solution that is consistent with another previously determined RNA G4 structure by SAXS [31]. Interestingly, the DNA equivalent (Q2DNA) presented markedly different features, namely in terms of its CD profile and its inability to stain with a parallel G4 dye, despite adopting a similar shape in solution as determined by SAXS and sharing similar hydrodynamic features as Q2RNA. Although a high-resolution structure would unambiguously highlight the differences, our data is consistent with Q2DNA adopting the hybrid-type G4 orientation observed in group II G4s, which has three parallel and one antiparallel strands [55][56][57]. We anticipate that the ability to accommodate 2 0 - OH groups in RNA G4s (affecting hydrogen bonding and sugar puckering) is central to the observed conformational differences between Q2RNA and Q2DNA [61]. Previous low-resolution structural and biophysical studies suggest that the N-terminal domain of RHAU interacts with the G-quartet face on the top or bottom plane of the G4 for both RNA and DNA [31]. Recently, a high-resolution structure of a short N-terminal peptide in complex with a DNA G4 has reinforced this mode of recognition, but also suggested that certain basic amino acid residues mediate specificity through interaction with the phosphodiester backbone [30]. Recognition of the Q2RNA G4 by the N-terminal region of RHAU (containing the RSM) uses nearly identical amino acid residues to those previously observed as important with another RNA G4 [31]. The elongated solution structure of Q2RNA/RHAU 53-105 by SAXS is consistent with the same protein truncation in complex with another RNA G4 [31], and the superimposition of individual Q2RNA and RHAU 53-105 models onto the complex model are consistent with recognition of the G-quartet face as the primary site of recognition.
Mechanistic studies have also suggested the importance of a parallel orientation for the recognition by RHAU [24,30]. The parallel G4 specific dye used in this study (NMM) interacts by stacking on the G-tetrad faces [53] and we have observed a significant reduction in the staining intensity of the dye where the G4 is bound to RHAU 53-105 as opposed to free G4 (data not shown). This suggests that the protein occupies the tetrad face. A previous study investigating a G4 from the human telomerase RNA (hTR) and its DNA counterpart has revealed that both adopt a parallel orientation and that both interact with RHAU 53-105 by means of the RSM. However, the DNA G4 made additional interactions with RHAU that were not observed in the RNA G4 [31]. DNA G4s generally demonstrate lower affinity for RHAU than their RNA counterparts [28,30,31], and whether the 2'-OH, a parallel arrangement, or both are important remains to be determined for RNA binding. Given these observations, it was not surprising that different strand orientations adopted by Q2RNA and Q2DNA significantly impact their affinity for RHAU. In the absence of a high-resolution RHAU-RNA G4 structure, our results strongly support the previously observed mode of recognition where strand directionality is key to presenting a parallel G4 face for RHAU binding. High-resolution structural studies of RNA G4s in complex with RHAU will likely confirm the hypothesis that both electrostatic and steric impacts of the 2'-OH also fulfill an important role.
While the importance of the N-terminal domain of RHAU has clearly been established, the mechanism whereby full-length protein binds and unwinds G4 structures remains to be elucidated. Binding of truncated RHAU  to RNA or DNA G4 does not attain the full binding affinity observed in full-length RHAU nor does it induce unwinding [27,28,31]. These features are clearly confirmed again in this study as full-length RHAU has higher affinity than the Nterminal fragment for Q2RNA, and comparison of the CD spectra of Q2RNA free and in complex with RHAU  indicates no G4 unwinding. Therefore, future studies geared towards an understanding of G4 helicase activity in the context of the full-length protein, remain a priority. The work presented here, while focused specifically on the in vitro study of a purified RNAprotein complex, provide the template for an eventual mechanistic understanding of G4 impact on translational regulation of mRNAs, including PITX1.