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Article

RNA Binding Properties of SOX Family Members

by
Seyed Mohammad Ghafoori
1,†,
Ashish Sethi
2,3,†,
Gayle F. Petersen
4,†,
Mohammad Hossein Tanipour
2,
Paul R. Gooley
2 and
Jade K. Forwood
1,4,*
1
School of Dentistry and Medical Sciences, Charles Sturt University, Wagga Wagga, NSW 2678, Australia
2
Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3010, Australia
3
Australian Nuclear Science Technology Organisation, The Australian Synchrotron, 800 Blackburn Rd., Clayton, VIC 3168, Australia
4
Gulbali Institute, Charles Sturt University, Wagga Wagga, NSW 2678, Australia
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Cells 2024, 13(14), 1202; https://doi.org/10.3390/cells13141202
Submission received: 25 July 2023 / Revised: 9 July 2024 / Accepted: 11 July 2024 / Published: 16 July 2024
Browse Figures
Versions Notes

Abstract

:
SOX proteins are a family of transcription factors (TFs) that play critical functions in sex determination, neurogenesis, and chondrocyte differentiation, as well as cardiac, vascular, and lymphatic development. There are 20 SOX family members in humans, each sharing a 79-residue L-shaped high mobility group (HMG)-box domain that is responsible for DNA binding. SOX2 was recently shown to interact with long non-coding RNA and large-intergenic non-coding RNA to regulate embryonic stem cell and neuronal differentiation. The RNA binding region was shown to reside within the HMG-box domain; however, the structural details of this binding remain unclear. Here, we show that all SOX family members, except group H, interact with RNA. Our mutational experiments demonstrate that the disordered C-terminal region of the HMG-box domain plays an important role in RNA binding. Further, by determining a high-resolution structure of the HMG-box domain of the group H family member SOX30, we show that despite differences in RNA binding ability, SOX30 shares a very similar secondary structure with other SOX protein HMG-box domains. Together, our study provides insight into the interaction of SOX TFs with RNA.
Keywords:
SOX; HMG-box; RNA binding

1. Introduction

Sex-determining region Y (SRY) was the founding member of a 20-member family of transcription factors (TFs) known as SRY-related high mobility group (HMG)-box (SOX) proteins. SOX proteins play crucial roles in various biological processes, including development, organogenesis, cell fate, and homeostasis [1,2,3,4,5]. All SOX family members share a common 79-residue HMG-box domain, with >50% sequence similarity to the SRY HMG-box (Figure 1) [6]. The L-shaped HMG-box comprises three α-helices: α1 and α2 that form one arm of the L (major wing), and α3 that forms the other arm of the L (minor wing) [7]. The HMG-box is responsible for binding and bending DNA [8], specifically at the consensus site (A/T)(A/T)CAA(A/T)G [9]. Unlike most other TFs, the HMG-box of SOX proteins binds to the minor groove of DNA, inducing a bend of 60–70° due to a wedge formed by the conserved Phe-Met (FM) dipeptide positioned on α1 that intercalates between bases and kinks the DNA [10]. The SOX HMG-box also features three key regions for nuclear localisation: two basic regions for nuclear import at the distal ends of the HMG-box domain and one leucine-rich nuclear export signal [11,12,13,14]. These regions regulate nucleocytoplasmic trafficking of SOX proteins, resulting in varying subcellular distribution throughout development.
Based on phylogenetic analysis of the HMG-box domain, SOX proteins are divided into nine groups (A, B1, B2, C, D, E, F, G, and H). SRY is the only member of the SOXA group, with an essential role in sex determination [6]. SOXB1 group members (SOX1, SOX2, and SOX3) play important roles in neural development, specifically formation of the neural primordium [16], proliferation and differentiation of neural stem cells during embryogenesis [17], and regulation of the maintenance/proliferation of adult neural stem cells during neurogenesis [18], as well as lens development, eye morphogenesis [19,20,21], inner ear development, and sensory hair cell differentiation [22]. SOXB2 group members (SOX14 and SOX21) also play a role in neural differentiation, negatively repressing the downstream Notch signalling molecule HES5 to promote neurogenesis and differentiation of neural stem cells [23]. Members of the SOXC group (SOX4, SOX11, and SOX12) contribute to nervous system development [24] and retinal differentiation [25]. In addition to their role in neural development [26], SOXD group members (SOX5, SOX6, and SOX13) play a critical role in chondrocyte differentiation and cartilage formation [27,28]. The main function of SOXE group members (SOX8, SOX9, and SOX10) is sex determination [29], while SOXF group members (SOX7, SOX17, and SOX18) play a crucial role in cardiac, vascular, and lymphatic development [30]. Finally, the functions of SOXG and SOXH group members (SOX15 and SOX30, respectively) are not yet fully elucidated; however, they have been shown to play roles in cancer prevention and apoptosis [31,32].
It has been demonstrated that some TFs possess the ability to bind both DNA and RNA [33,34,35]. For instance, overexpression of p53 has been shown to suppress mdmx mRNA translation by binding to the 5′ untranslated region of the mdmx mRNA [36]. Similarly, Ubx can bind RNA, regulating mRNA expression and co-transcriptional splicing [37], and YY1 can bind gene regulatory elements and their associated RNA, contributing to the maintenance of some TFs at gene regulatory elements [34]. Recently, it was identified that SOX2 can also bind both DNA and RNA. RNA immunoprecipitation experiments demonstrated an association between SOX2 and long non-coding RNA (lncRNA)_ES1 (AK056826) and lncRNA_ES2 (EF565083) for regulation of embryonic stem cell (ESC) pluripotency [38]. A further study found that SOX2 binds to lncRNA_ES2 through its DNA-binding HMG-box, with both high affinity and low sequence specificity [39]. Studies have also shown that the lncRNA RMST associates with SOX2 and regulates neuronal differentiation [40], and the large-intergenic non-coding RNA 1614 interacts with SOX2 to mediate transcriptional silencing and maintain ESC pluripotency [41]. One study has demonstrated that Sox2 binds RNA via a 60 amino acid region directly after the HMG-box, with a preference for GC-rich RNA sequences [42], whereas another study has linked the RNA binding ability of TFs, including SOX2, to an Arginine Rich Motif (ARM)-like domain encompassing the C-terminal end of the HMG-box and residues directly after this [43]. Due to a lack of structural and molecular information detailing how SOX proteins bind RNA, here we examine the RNA binding properties across a range of SOX family members and demonstrate that binding resides in the HMG-box C-terminal region.

2. Materials and Methods

2.1. Protein Expression and Purification

The HMG-box domains from one member of each SOX group were cloned into different vectors (Table S1) and transformed into BL21 (DE3) pLysS E. coli cells (ThermoFisher Scientific, Waltham, MA, USA) for protein expression. Cells were grown in 5 mL of Luria–Bertani (LB) media (tryptone 10 g/L, yeast extract 5 g/L, sodium chloride 10 g/L) supplemented with the appropriate antibiotic at 37 °C until the OD600 reached 0.6–0.8. For large-scale protein expression, 1 mL of starter culture was added to 1 L of expression media (tryptone 10 g/L, yeast extract 5 g/L, dipotassium hydrogen phosphate 8.7 g/L, potassium dihydrogen phosphate 6.8 g/L, sodium sulphate 0.71 g/L, magnesium sulphate 0.24 g/L, glycerol 5 g/L, glucose 0.5 g/L, lactose 2 g/L) with the appropriate antibiotic, and expression was induced for 36 h at room temperature using the auto-induction method described previously [44]. For the SOX6 HMG-box domain, expression was induced for 24 h at 18 °C using the IPTG induction method described previously [45], using IPTG at 1 mM. Cells were harvested at 6400 RCF for 20 min and resuspended in low imidazole phosphate buffer (50 mM phosphate buffer pH 8.0, 300 mM sodium chloride, 20 mM imidazole). Prior to purification, cells were lysed using three freeze–thaw cycles [46] and treatment with 0.5 mg DNaseI (Sigma-Aldrich, St. Louis, MO, USA) and 20 mg lysozyme (Sigma-Aldrich, St. Louis, MO, USA) for 45 min at room temperature. Lysate was injected onto a HisTrap 5 mL column (Cytiva, Marlborough, MA, USA) using low imidazole phosphate buffer, followed by washing with 15 column volumes (CVs) of the same buffer. The sample was eluted with high imidazole phosphate buffer (50 mM phosphate buffer pH 8.0, 300 mM sodium chloride, 500 mM imidazole) using a gradient elution for 5 CVs, followed by 5 CVs of 100% high imidazole phosphate buffer. Eluted fractions were pooled and split into three tubes, of which one was treated with 0.5 mg DNaseI, one was treated with 0.5 mg RNaseA (Sigma-Aldrich, St. Louis, MO, USA), and one was left untreated, prior to incubation at 4 °C on a roller overnight. Analytical gel filtration was performed with 1 mL of each sample on a Superdex 200 pg 10/300 GL column (Cytiva, Marlborough, MA, USA) using gel filtration buffer (50 mM tris, 125 mM sodium chloride, pH 8.0).

2.2. Protein/RNA Characterisation

Select analytical gel filtration peak fractions were run on precast 4–12% polyacrylamide Bis-Tris gels (ThermoFisher Scientific, Waltham, MA, USA) using Bolt MES SDS Running Buffer (ThermoFisher Scientific, Waltham, MA, USA) (to visualise protein) and 1.5% agarose gels containing GelRed (Sigma-Aldrich, St. Louis, MO, USA) (1 μL/100 mL) using tris-boric acid (TB) buffer (45 mM tris, 45 mM boric acid), pH 8.5 (to visualise RNA). Polyacrylamide gels were stained with Coomassie blue (0.2% Coomassie brilliant blue, 10% ethanol, 10% glacial acetic acid) and destained (10% ethanol, 10% glacial acetic acid) overnight. Gels were imaged using a Bio-Rad Gel Doc XR+ Imaging System (Bio-Rad Laboratories, Hercules, CA, USA) and images were processed using Image Lab Software (version 6.0.1, Bio-Rad Laboratories, Hercules, CA, USA) and Adobe Photoshop (version 24.0, Adobe, San Jose, CA, USA).

2.3. Electrophoretic Mobility Shift Assay (EMSA)

Select ssDNA (Integrated DNA Technologies, Coralville, IA, USA; Table S2) (10 μL of 100 μM) were mixed with SOX proteins (10 μL of 100 μM) and incubated at room temperature for 15 min. Samples were supplemented with 5 μL of 50% glycerol and run on a 1.5% agarose gel containing GelRed (1 μL/100 mL) for 75 min at 70 V in TB buffer, pH 7.4. The gel was imaged, stained with Coomassie blue, destained, and imaged again. Gels were imaged using a Bio-Rad Gel Doc XR+ Imaging System and images were processed and colour-edited using Adobe Photoshop [46].

2.4. Fluorescence Polarisation

Two-fold serial dilutions of 20 µM SOX proteins (RNAse-treated) were titrated across 23 wells of a black Fluotrac microplate (Greiner Bio-One, Kremsmünster, Austria) and incubated with 80 nM 3′ FAM-labelled RNA (Integrated DNA Technologies, Coralville, IA, USA; Table S2). Wells were made up to a total volume of 200 µL with gel filtration buffer and fluorescence polarisation was measured using a CLARIOstar Plus plate reader (BMG Labtech, Ortenberg, Germany). Assays were performed in triplicate and included a no protein control used for gain adjustment. Data were analysed in GraphPad Prism (version 10.2.2, GraphPad, San Diego, CA, USA) using non-linear regression assuming one site-specific binding.

2.5. Nuclear Magnetic Resonance (NMR)

2.5.1. Expression and Purification

The SOX17 HMG-box domain was expressed in BL21 (DE3) pLysS E. coli cells using the autoinduction method [44]. For labelling with 13C and 15N isotopes, cells were grown in N-5052 [47] supplemented with 3 g/L of D-[13C] glucose (Sigma-Aldrich, St. Louis, MO, USA) and 1 g/L of 15NH4Cl (Sigma-Aldrich, St. Louis, MO, USA) as the sole sources of carbon and nitrogen, respectively. Cells were grown at 37 °C to an OD600 of 0.6–0.7, transferred to 16 °C, and induced (0.4 mM IPTG with shaking overnight at 225–230 rpm). Protein was purified as above, and stored at −80 °C for future use.

2.5.2. NMR Spectroscopy

NMR experiments were performed at 25 °C on a 700 MHz Bruker Avance HDIII spectrometer (Bruker, Billerica, MA, USA) equipped with a triple resonance cryoprobe, using protein dissolved in size exclusion chromatography (SEC) buffer (50 mM tris, 300 mM sodium chloride, 7 mM DTT, pH 8.0). Backbone resonances (13Cα, 13Cβ, 13C’, 15N, and NH) of residues were assigned from 3D HNCACB, HN(CO)CACB, HNCO, and HN(CA)CO experiments acquired using non-uniform sampling (NUS). For NUS, sampling schedules were generated using Poisson-gap sampling with 10% of the total number of points collected for all 3D NMR experiments [48]. Spectra were reconstructed with compressed sensing algorithms using qMDD [49] and processed using NMRPipe [50], and data were analysed in NMRFAM-SPARKY [51]. To monitor binding of a 12-mer ssDNA (Integrated DNA Technologies, Coralville, IA, USA; Table S2) to SOX17, 2D 15N,1H Heteronuclear Single Quantum Coherence (HSQC)-monitored titrations (2048 × 256 data points) were conducted using 100 µM of 15N-labelled SOX17 with an increasing concentration (25–100 µM) of 12-mer ssDNA. During titrations, the NMR sample volume was kept within a variation of 10%. The average chemical change was determined from Δδ ppm = √[(Δδ1HN)2 + (0.15 × Δδ15N)2] [52].

2.6. Crystallisation and Structure Determination

The SOX30 HMG-box domain was cloned, expressed, and purified using nickel affinity chromatography, as described above. Fractions were pooled and further purified by SEC on a Superdex 200 pg 26/600 column (Cytiva, Marlborough, MA, USA) using SEC buffer. Protein was concentrated using an Amicon 10 kDa molecular weight cutoff centrifugal filter (Merck Millipore, Burlington, MA, USA) to 31 mg/mL, aliquoted, and stored at −80 °C. Crystals were produced using the hanging drop vapour diffusion method over 300 μL of reservoir solution. Needle-shaped crystals formed in 0.1 M sodium acetate, 2 M ammonium sulphate, pH 4.6, in 5–7 days. X-ray diffraction data were collected at the Australian Synchrotron on the MX2 beamline using an Eiger 16M detector. iMosflm was used for data reduction and integration [53]. Aimless was used for merging, space group assignment, and scaling, with selection of 5% reflections for Rfree calculations [54]. PhaserMR was used for molecular replacement using PDB ID: 1O4X as the search model [55], and Phenix was used for refinement [56]. Coot was used for modelling [57]. The final model has been validated and deposited in the Protein Data Bank with PDB ID: 7JJK.

3. Results and Discussion

3.1. RNA Binding Properties of SOX Proteins Extend to All Family Members Except Group H

Based on reports that SOX2 binds both DNA and RNA through its HMG-box domain [39], we investigated whether this RNA binding property extends to other SOX family members. The HMG-box domains of representative SOX proteins from each of the nine groups (Figure 1) were cloned, expressed, purified, and tested for their ability to bind RNA. Our initial assay relied on the ability of SOX HMG-box proteins to co-purify with nucleic acid. Following affinity purification, SOX proteins were either left untreated or treated with DNase or RNase, before further purification on an analytical gel filtration column. Fractions were analysed by both SDS-PAGE and agarose gel electrophoresis.
We found that the SOX17 HMG-box domain (group F) co-purified with a large amount of RNA (Figure 2A). While some of the SOX17:RNA complex dissociated during analytical gel filtration, a proportion of SOX17 co-eluted with RNA. Treatment with DNase and RNase confirmed that the majority of the bound nucleic acid was RNA, since treatment with RNase removed most of the absorbance associated with fractions 9–16 and shifted the RNA peak towards the end of the elution profile, indicative of digested RNA, and resulted in the least nucleic acids visible on the agarose gels. The majority of the absorbance on the analytical gel filtration profiles could be attributed to RNA, based on the large second peak that appeared upon RNase treatment which was almost three times greater than the peak associated with SOX17. Experiments performed with SRY (group A), SOX2 (group B1), SOX21 (group B2), SOX11 (group C), SOX6 (group D), SOX9 (group E), and SOX15 (group G) HMG-box domains all similarly co-purified with RNA (Figures S1–S7). Interestingly, we found that the SOX30 HMG-box domain (group H) did not co-purify with any RNA, with all analytical gel filtration profiles appearing very similar for no treatment, RNase-treated, and DNase-treated samples, as well as the absence of any detectable nucleic acid in the agarose gels (Figure 2B).
In summary, representative SOX proteins from each group, with the exception of group H (SOX30), bound RNA. While some SOX proteins bound large amounts of RNA, to the extent that the RNA peak on the analytical gel filtration profile surpassed that of the protein following RNase treatment (SRY [group A], SOX2 [group B1], SOX21 [group B2], SOX11 [group C], SOX17 [group F], and SOX15 [group G]), others bound smaller amounts of RNA, with a greater protein peak than RNA peak (SOX6 [group D] and SOX9 [group E]). Finally, SOX30 (group H) showed no affinity for RNA.

3.2. The SOX HMG-Box Domain Interacts with ssDNA

To further examine the RNA binding properties of SOX family members and establish whether there is a direct binding interaction, each of the SOX proteins were purified free of nucleic acids by nuclease treatment and subsequent purification steps. To confirm that all purified protein was free of nucleic acids, we ran the protein alone on an agarose gel, as well as spectrophotometrically confirmed the presence of pure protein using an absorbance ratio of 260/280, with a value of 0.7 indicating pure protein and a protein/RNA mixture typically with values of 1.7. We then tested whether these proteins were able to bind a 60-mer ssDNA nucleic acid probe via EMSA (Figure 3A). We found that both SRY and the probe shifted and co-migrated, indicating direct binding of the SRY:60-mer complex. Both SOX2 and SOX21 also shifted the probe, indicating direct binding; however, these SOX:60-mer complexes failed to migrate from the well, potentially due to decreased solubility upon complex formation. SOX6, SOX9, SOX11, SOX15, and SOX17 all exhibited altered migration paths of the protein and the probe, similarly indicating direct binding. In agreement with our observation that SOX30 failed to bind RNA, SOX30 was unable to alter migration of the probe, indicating no direct binding. To validate what was shown with ssDNA, fluorescence polarisation was utilised to measure binding affinity between SOX proteins and a FAM-labelled RNA probe previously shown to bind SOX2 [39]. SOX2 bound RNA with high affinity (Kd ~57 nM), consistent with previous reports [39]. Compared to the SOX2 control, SOX17 (representative RNA binding SOX protein) bound RNA with ~6-fold weaker affinity at a Kd of ~327 nM, while no RNA binding was detected for SOX30 (Figure 3B).

3.3. The C-Terminal Region of the SOX17 HMG-Box Domain Is Responsible for RNA Interaction

To identify the regions responsible for RNA interaction, we performed crystallographic and NMR experiments with the SOX17 HMG-box domain, which was selected due to its obvious RNA/ssDNA binding ability, as demonstrated in the analytical gel filtration purification, EMSA, and fluorescence polarisation data. Whilst the crystallographic approach failed to produce diffracting crystals, NMR was able to identify key shifts in the 15N,1H HSQC spectra of 15N-labelled SOX17 upon titration (1:1) with a 12-mer ssDNA nucleic acid probe (Figure 4). Significant chemical shift changes were observed for the C-terminal region (Arg125 to Arg138), the N-terminal region (Ile68, Ala74, and Met76), and residues in the central helix (Glu97 and Lys100). The indole signal of Trp106 also shifted and significantly broadened on titration with the probe.
Due to the large number of chemical shift changes in the N- and C-terminal regions, we designed a series of mutants with N- and/or C-terminal truncations (denoted as ∆) of the SOX17 HMG-box domain (Figure 5; Table S3), only removing residues outside of the α-helices. We found that while wild-type (WT) SOX17 and SOX17 ∆N bound RNA via analytical gel filtration and were able to shift migration of a ssDNA nucleic acid probe, SOX17 ∆C and SOX17 ∆CN mutants were unable to (Figure 5A,B). We also measured binding affinity between SOX17 truncation proteins and a FAM-labelled RNA probe, with SOX17 ∆C and SOX17 ∆CN abolishing RNA binding compared to SOX17 WT. Some RNA binding was detected at the highest concentrations of SOX17 ∆N; however, the binding affinity was too low to be determined (Figure 5C). This further indicates the importance of the C-terminal region, specifically the seven amino acid region 138-RPRRRKQ-144 (73–79 HMG-box numbering), in the RNA binding ability of the SOX HMG-box domain. To investigate whether these deletions could similarly affect other SOX family members, we also assessed whether SRY ∆C, SOX2 ∆C, and SOX11 ∆C mutants were able to bind RNA, finding that in all cases, removal of the seven amino acid C-terminal region (73–79 HMG-box numbering) prevented RNA binding (Figure 6).

3.4. SOX30 Retains a Structured HMG-Box Domain

Due to its inability to bind RNA, we sought to characterise the structure of the SOX30 HMG-box domain. The protein was recombinantly expressed, purified, and crystallised. Crystals formed in 0.1 M sodium acetate, 2 M ammonium sulphate, pH 4.6, and diffracted to 1.4 Å resolution. The diffraction data were indexed and integrated in the space group P212121. The structure was solved by molecular replacement in Phaser using the α-helices of SOX2 as the reference model (PDB ID: 1O4X) [55], followed by rebuilding in COOT and refinement in Phenix [56] (see Table 1 for data collection and refinement statistics). The structure was deposited to the Protein Data Bank with PDB ID: 7JJK.
The crystal structure revealed that the SOX30 HMG-box domain contains the typical features of an HMG-box, including three α-helices and two disordered regions towards the N- and C-termini. The three α-helices form an L-shape in which α1 and α2 create the major wing and α3 makes the minor wing (Figure 7A). Superimposing our SOX30 HMG-box domain structure on other available SOX protein HMG-box domain structures (alone and DNA-bound) demonstrated a very similar secondary structure between SOX family members (Figure 7B), with a low RMSD (Table 2). The largest differences between the superimposed SOX structures are observed at the N- and C-termini, which is to be expected given that these regions are disordered and adopt multiple conformations. The C-terminal end of the SOX30 structure is seen to be orientated in a different direction to the other SOX structures. However, this is due to differences in crystal packing, with the SOX30 molecule within the asymmetric unit sandwiched between two adjacent SOX30 molecules, forming crystal contacts that stabilise the C-terminal end in this conformation. The seven amino acids at the C-terminal end of the HMG-box that we identified as critical for RNA binding (407-QPRPGKR-413; 73–79 HMG-box numbering) are not visible in the SOX30 structure. This critical binding region is located within the disordered C-terminal end of the HMG-box domain, and thus is often not visible in structures, including those of Sox5, SOX9, and SOX17. Regardless, the structural similarities in the remainder of the SOX30 HMG-box support our claim that this C-terminal end is key for RNA binding.
Structurally, the HMG-box domain of SOX30 retained all of the key features of other SOX proteins, with no obvious structural differences that would indicate why SOX30 does not bind RNA. Inspection of the C-terminal region of the SOX30 HMG-box domain, shown here to be responsible for RNA binding in other SOX family members, revealed sequence differences that are distinct from other SOX proteins. As shown in Figure 1, the final five C-terminal residues in the HMG-box domain (residues 75–79, HMG-box numbering) feature a consensus sequence of Rrkkk, thus containing a strong clustering of positive residues. Conversely, SOX30 has the sequence RPGKR, and thus has lost 40% of its positive charge within this cluster, which may contribute to the loss of RNA binding ability.
In the present study, we demonstrate that the HMG-box domains of representatives of all SOX groups, with the exception of SOX30 (group H), bind RNA. While the DNA binding capability and function of SOX proteins have been well characterised, including detailed structural approaches [59,60,61,62,63,65], little is known as to how SOX proteins interact with RNA. Here, we show that the disordered C-terminal region of the HMG-box domain of SOX proteins is critical for RNA binding. Our NMR studies indicate that although chemical shifts can be observed across a range of residues within the HMG-box of SOX17, the shift is significantly greater in the basic-rich C-terminal region. Consistent with SOX17 data, we also show that C-terminal truncation of the HMG-box domains of SRY, SOX2, and SOX11 result in a dramatic reduction in RNA binding.
The consensus sequence of the C-terminal tail of the HMG-box domain of SOX proteins (70-ykYrPRrkkk-79, Figure 1) may provide insights into the differences in RNA binding ability of SOX proteins. SOX family members that bound larger amounts of RNA (SRY, SOX2, SOX21, SOX11, SOX17, and SOX15) feature the common sequence 70-YKYRPRR/K-76, whereas SOX6 and SOX9 that bound smaller amounts of RNA have the sequences 70-YKYKPRP-76 and 70-YKYQPRR-76, respectively. Conversely, SOX30, which shows no RNA binding, features the sequence 70-WVYQPRP-76. As the PR motif (74–75) is conserved throughout all SOX proteins, it is not the sole RNA-binding determinant. The R/K residues flanking this motif (73/76) enhance, but are not strictly required for, RNA binding, as group D and E SOX proteins lack these residues yet still (weakly) bind RNA. Residues 70-YKY-72 upstream of the PR motif are conserved in all SOX proteins except for SOX30 and may influence RNA binding; however, they are also not the sole binding determinant as △C truncations retaining these residues still lost RNA binding ability. In addition, SOX30 is the only protein lacking a basic residue at position 77 (K/R > G). As such, it is likely that multiple residues within the HMG-box domain C-terminus are required to confer RNA binding.
A 60-amino acid region directly after the HMG-box has previously been linked to the RNA binding ability of Sox2 [42,43]. This differs from our data that identify a seven amino acid region at the C-terminal end of the SOX protein HMG-box (73-rPRrkkk-79; Figure 1, black dashed box) as being critical for RNA binding; however, our study was restricted to the HMG-box only and did not investigate regions outside of this. Another study linked the RNA binding ability of SOX2 to an ARM-like domain encompassing the C-terminal end of the HMG-box and residues directly after this, with EMSA analysis of RNA with a peptide encoding R/K > A mutations of the SOX2-ARM demonstrating abolished RNA binding [43]. The region identified in this study includes the seven amino acids we found to be critical for RNA binding, confirming the importance of basic residues in this region for RNA binding of SOX proteins. This region found to be necessary for RNA binding has also been shown to be involved in DNA binding, as well as interactions with importins that drive nuclear import [14]. Whilst speculative, this competition between important cellular binding partners may play a role in the ability of SOX proteins to differentially regulate development over a wide range of cell and tissue types. While further detailed experiments will be required to elucidate this, our study provides an important insight into the regions that can be targeted to dissect such interactions and the important biological functions of SOX proteins.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/cells13141202/s1, Figure S1: The SRY HMG-box domain co-purifies with RNA during affinity and size exclusion chromatography; Figure S2: The SOX2 HMG-box domain co-purifies with RNA during affinity and size exclusion chromatography; Figure S3: The SOX21 HMG-box domain co-purifies with RNA during affinity and size exclusion chromatography; Figure S4: The SOX11 HMG-box domain co-purifies with RNA during affinity and size exclusion chromatography; Figure S5: The SOX6 HMG-box domain co-purifies with RNA during affinity and size exclusion chromatography; Figure S6: The SOX9 HMG-box domain co-purifies with RNA during affinity and size exclusion chromatography; Figure S7: The SOX15 HMG-box domain co-purifies with RNA during affinity and size exclusion chromatography; Table S1: SOX HMG-box domain constructs; Table S2: Nucleic acid binding substrate sequences; Table S3: SOX HMG-box domain mutant constructs.

Author Contributions

Conceptualization, S.M.G. and J.K.F.; Methodology, S.M.G., A.S. and G.F.P.; Formal Analysis, S.M.G., A.S., G.F.P. and P.R.G.; Investigation, S.M.G., A.S., G.F.P. and M.H.T.; Resources, P.R.G. and J.K.F.; Data Curation, S.M.G., A.S. and G.F.P.; Writing—Original Draft Preparation, S.M.G., A.S., G.F.P., M.H.T., P.R.G. and J.K.F.; Writing—Review and Editing, S.M.G., G.F.P. and J.K.F.; Visualisation, S.M.G., A.S. and G.F.P.; Supervision, P.R.G. and J.K.F.; Project Administration, S.M.G.; Funding Acquisition, J.K.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Health and Medical Research Council grant number APP1188175 awarded to J.K.F.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Files associated with the structure generated in this study have been deposited to the Protein Data Bank and were released prior to submission of the manuscript with PDB ID: 7JJK. Source data are provided with the paper.

Acknowledgments

This research was undertaken in part using the MX2 beamline at the Australian Synchrotron, part of ANSTO, and made use of the Australian Cancer Research Foundation (ACRF) detector. The authors thank Jeff Nanson for constructive criticism of the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Gubbay, J.; Collignon, J.; Koopman, P.; Capel, B.; Economou, A.; Münsterberg, A.; Vivian, N.; Goodfellow, P.; Lovell-Badge, R. A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embryonically expressed genes. Nature 1990, 346, 245–250. [Google Scholar] [CrossRef] [PubMed]
  2. Sinclair, A.H.; Berta, P.; Palmer, M.S.; Hawkins, J.R.; Griffiths, B.L.; Smith, M.J.; Foster, J.W.; Frischauf, A.-M.; Lovell-Badge, R.; Goodfellow, P.N. A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 1990, 346, 240–244. [Google Scholar] [CrossRef] [PubMed]
  3. Kamachi, Y.; Uchikawa, M.; Kondoh, H. Pairing SOX off: With partners in the regulation of embryonic development. Trends Genet. 2000, 16, 182–187. [Google Scholar] [CrossRef] [PubMed]
  4. Kamachi, Y.; Uchikawa, M.; Tanouchi, A.; Sekido, R.; Kondoh, H. Pax6 and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens development. Genes Dev. 2001, 15, 1272–1286. [Google Scholar] [CrossRef]
  5. Sarkar, A.; Hochedlinger, K. The Sox family of transcription factors: Versatile regulators of stem and progenitor cell fate. Cell Stem Cell 2013, 12, 15–30. [Google Scholar] [CrossRef]
  6. She, Z.-Y.; Yang, W.-X. SOX family transcription factors involved in diverse cellular events during development. Eur. J. Cell Biol. 2015, 94, 547–563. [Google Scholar] [CrossRef] [PubMed]
  7. Weiss, M.A. Floppy SOX: Mutual induced fit in HMG (high-mobility group) box-DNA recognition. Mol. Endocrinol. 2001, 15, 353–362. [Google Scholar] [CrossRef] [PubMed]
  8. van Beest, M.; Dooijes, D.; van de Wetering, M.; Kjaerulff, S.; Bonvin, A.; Nielsen, O.; Clevers, H. Sequence-specific high mobility group box factors recognize 10–12-base pair minor groove motifs. J. Biol. Chem. 2000, 275, 27266–27273. [Google Scholar] [CrossRef]
  9. Wegner, M. All purpose Sox: The many roles of Sox proteins in gene expression. Int. J. Biochem. Cell Biol. 2010, 42, 381–390. [Google Scholar] [CrossRef]
  10. Hou, L.; Srivastava, Y.; Jauch, R. Molecular basis for the genome engagement by Sox proteins. Semin. Cell Dev. Biol. 2017, 63, 2–12. [Google Scholar] [CrossRef]
  11. Malki, S.; Boizet-Bonhoure, B.; Poulat, F. Shuttling of SOX proteins. Int. J. Biochem. Cell Biol. 2010, 42, 411–416. [Google Scholar] [CrossRef]
  12. Gasca, S.; Cañizares, J.; de Santa Barbara, P.; Méjean, C.; Poulat, F.; Berta, P.; Boizet-Bonhoure, B. A nuclear export signal within the high mobility group domain regulates the nucleocytoplasmic translocation of SOX9 during sexual determination. Proc. Natl. Acad. Sci. USA 2002, 99, 11199–11204. [Google Scholar] [CrossRef]
  13. Südbeck, P.; Scherer, G. Two independent nuclear localization signals are present in the DNA-binding high-mobility group domains of SRY and SOX9. J. Biol. Chem. 1997, 272, 27848–27852. [Google Scholar] [CrossRef]
  14. Jagga, B.; Edwards, M.; Pagin, M.; Wagstaff, K.M.; Aragão, D.; Roman, N.; Nanson, J.D.; Raidal, S.R.; Dominado, N.; Stewart, M.; et al. Structural basis for nuclear import selectivity of pioneer transcription factor SOX2. Nat. Commun. 2021, 12, 28. [Google Scholar] [CrossRef]
  15. Okonechnikov, K.; Golosova, O.; Fursov, M.; Team, U. Unipro UGENE: A unified bioinformatics toolkit. Bioinformatics 2012, 28, 1166–1167. [Google Scholar] [CrossRef] [PubMed]
  16. Pevny, L.; Placzek, M. SOX genes and neural progenitor identity. Curr. Opin. Neurobiol. 2005, 15, 7–13. [Google Scholar] [CrossRef]
  17. Graham, V.; Khudyakov, J.; Ellis, P.; Pevny, L. SOX2 functions to maintain neural progenitor identity. Neuron 2003, 39, 749–765. [Google Scholar] [CrossRef]
  18. Episkopou, V. SOX2 functions in adult neural stem cells. Trends Neurosci. 2005, 28, 219–221. [Google Scholar] [CrossRef]
  19. Matsushima, D.; Heavner, W.; Pevny, L.H. Combinatorial regulation of optic cup progenitor cell fate by SOX2 and PAX6. Development 2011, 138, 443–454. [Google Scholar] [CrossRef]
  20. Langer, L.; Taranova, O.; Sulik, K.; Pevny, L. SOX2 hypomorphism disrupts development of the prechordal floor and optic cup. Mech. Dev. 2012, 129, 1–12. [Google Scholar] [CrossRef]
  21. Nishiguchi, S.; Wood, H.; Kondoh, H.; Lovell-Badge, R.; Episkopou, V. Sox1 directly regulates the γ-crystallin genes and is essential for lens development in mice. Genes Dev. 1998, 12, 776–781. [Google Scholar] [CrossRef] [PubMed]
  22. Kiernan, A.E.; Pelling, A.L.; Leung, K.K.; Tang, A.S.; Bell, D.M.; Tease, C.; Lovell-Badge, R.; Steel, K.P.; Cheah, K.S. Sox2 is required for sensory organ development in the mammalian inner ear. Nature 2005, 434, 1031–1035. [Google Scholar] [CrossRef] [PubMed]
  23. Matsuda, S.; Kuwako, K.-i.; Okano, H.J.; Tsutsumi, S.; Aburatani, H.; Saga, Y.; Matsuzaki, Y.; Akaike, A.; Sugimoto, H.; Okano, H. Sox21 promotes hippocampal adult neurogenesis via the transcriptional repression of the Hes5 gene. J. Neurosci. 2012, 32, 12543–12557. [Google Scholar] [CrossRef] [PubMed]
  24. Wang, Y.; Lin, L.; Lai, H.; Parada, L.F.; Lei, L. Transcription factor Sox11 is essential for both embryonic and adult neurogenesis. Dev. Dynam. 2013, 242, 638–653. [Google Scholar] [CrossRef]
  25. Usui, A.; Iwagawa, T.; Mochizuki, Y.; Iida, A.; Wegner, M.; Murakami, A.; Watanabe, S. Expression of Sox4 and Sox11 is regulated by multiple mechanisms during retinal development. FEBS Lett. 2013, 587, 358–363. [Google Scholar] [CrossRef] [PubMed]
  26. Batista-Brito, R.; Rossignol, E.; Hjerling-Leffler, J.; Denaxa, M.; Wegner, M.; Lefebvre, V.; Pachnis, V.; Fishell, G. The cell-intrinsic requirement of Sox6 for cortical interneuron development. Neuron 2009, 63, 466–481. [Google Scholar] [CrossRef]
  27. Ikegami, D.; Akiyama, H.; Suzuki, A.; Nakamura, T.; Nakano, T.; Yoshikawa, H.; Tsumaki, N. Sox9 sustains chondrocyte survival and hypertrophy in part through Pik3ca-Akt pathways. Development 2011, 138, 1507–1519. [Google Scholar] [CrossRef] [PubMed]
  28. Nagy, A.; Kénesi, E.; Rentsendorj, O.; Molnár, A.; Szénási, T.; Sinkó, I.; Zvara, Á.; Thottathil Oommen, S.; Barta, E.; Puskás, L.G.; et al. Evolutionarily conserved, growth plate zone-specific regulation of the matrilin-1 promoter: L-Sox5/Sox6 and Nfi factors bound near TATA finely tune activation by Sox9. Mol. Cell. Biol. 2011, 31, 686–699. [Google Scholar] [CrossRef] [PubMed]
  29. Kashimada, K.; Koopman, P. Sry: The master switch in mammalian sex determination. Development 2010, 137, 3921–3930. [Google Scholar] [CrossRef]
  30. Hosking, B.; François, M.; Wilhelm, D.; Orsenigo, F.; Caprini, A.; Svingen, T.; Tutt, D.; Davidson, T.; Browne, C.; Dejana, E.; et al. Sox7 and Sox17 are strain-specific modifiers of the lymphangiogenic defects caused by Sox18 dysfunction in mice. Development 2009, 136, 2385–2391. [Google Scholar] [CrossRef]
  31. Han, F.; Liu, W.; Jiang, X.; Shi, X.; Yin, L.; Ao, L.; Cui, Z.; Li, Y.; Huang, C.; Cao, J.; et al. SOX30, a novel epigenetic silenced tumor suppressor, promotes tumor cell apoptosis by transcriptional activating p53 in lung cancer. Oncogene 2015, 34, 4391–4402. [Google Scholar] [CrossRef]
  32. Ding, Y.; Feng, Y.; Huang, Z.; Zhang, Y.; Li, X.; Liu, R.; Li, H.; Wang, T.; Ding, Y.; Jia, Z.; et al. SOX15 transcriptionally increases the function of AOC1 to modulate ferroptosis and progression in prostate cancer. Cell Death Dis. 2022, 13, 673. [Google Scholar] [CrossRef] [PubMed]
  33. Cassiday, L.A.; Maher, L.J., III. Having it both ways: Transcription factors that bind DNA and RNA. Nucleic Acids Res. 2002, 30, 4118–4126. [Google Scholar] [CrossRef] [PubMed]
  34. Sigova, A.A.; Abraham, B.J.; Ji, X.; Molinie, B.; Hannett, N.M.; Guo, Y.E.; Jangi, M.; Giallourakis, C.C.; Sharp, P.A.; Young, R.A. Transcription factor trapping by RNA in gene regulatory elements. Science 2015, 350, 978–981. [Google Scholar] [CrossRef]
  35. Castello, A.; Fischer, B.; Frese, C.K.; Horos, R.; Alleaume, A.-M.; Foehr, S.; Curk, T.; Krijgsveld, J.; Hentze, M.W. Comprehensive identification of RNA-binding domains in human cells. Mol. Cell 2016, 63, 696–710. [Google Scholar] [CrossRef] [PubMed]
  36. Tournillon, A.; Lopez, I.; Malbert-Colas, L.; Findakly, S.; Naski, N.; Olivares-Illana, V.; Karakostis, K.; Vojtesek, B.; Nylander, K.; Fåhraeus, R. p53 binds the mdmx mRNA and controls its translation. Oncogene 2017, 36, 723–730. [Google Scholar] [CrossRef] [PubMed]
  37. Carnesecchi, J.; Boumpas, P.; van Nierop y Sanchez, P.; Domsch, K.; Pinto, H.D.; Borges Pinto, P.; Lohmann, I. The Hox transcription factor Ultrabithorax binds RNA and regulates co-transcriptional splicing through an interplay with RNA polymerase II. Nucleic Acids Res. 2022, 50, 763–783. [Google Scholar] [CrossRef] [PubMed]
  38. Ng, S.-Y.; Johnson, R.; Stanton, L.W. Human long non-coding RNAs promote pluripotency and neuronal differentiation by association with chromatin modifiers and transcription factors. EMBO J. 2012, 31, 522–533. [Google Scholar] [CrossRef] [PubMed]
  39. Holmes, Z.E.; Hamilton, D.J.; Hwang, T.; Parsonnet, N.V.; Rinn, J.L.; Wuttke, D.S.; Batey, R.T. The Sox2 transcription factor binds RNA. Nat. Commun. 2020, 11, 1805. [Google Scholar] [CrossRef]
  40. Ng, S.-Y.; Bogu, G.K.; Soh, B.S.; Stanton, L.W. The long noncoding RNA RMST interacts with SOX2 to regulate neurogenesis. Mol. Cell 2013, 51, 349–359. [Google Scholar] [CrossRef]
  41. Guo, X.; Wang, Z.; Lu, C.; Hong, W.; Wang, G.; Xu, Y.; Liu, Z.; Kang, J. LincRNA-1614 coordinates Sox2/PRC2-mediated repression of developmental genes in pluripotency maintenance. J. Mol. Cell Biol. 2018, 10, 118–129. [Google Scholar] [CrossRef] [PubMed]
  42. Hou, L.; Wei, Y.; Lin, Y.; Wang, X.; Lai, Y.; Yin, M.; Chen, Y.; Guo, X.; Wu, S.; Zhu, Y.; et al. Concurrent binding to DNA and RNA facilitates the pluripotency reprogramming activity of Sox2. Nucleic Acids Res. 2020, 48, 3869–3887. [Google Scholar] [CrossRef]
  43. Oksuz, O.; Henninger, J.E.; Warneford-Thomson, R.; Zheng, M.M.; Erb, H.; Vancura, A.; Overholt, K.J.; Hawken, S.W.; Banani, S.F.; Lauman, R.; et al. Transcription factors interact with RNA to regulate genes. Mol. Cell 2023, 83, 2449–2463. [Google Scholar] [CrossRef] [PubMed]
  44. Roman, N.; Christie, M.; Swarbrick, C.M.; Kobe, B.; Forwood, J.K. Structural characterisation of the nuclear import receptor importin alpha in complex with the bipartite NLS of Prp20. PLoS ONE 2013, 8, e82038. [Google Scholar] [CrossRef] [PubMed]
  45. Sivashanmugam, A.; Murray, V.; Cui, C.; Zhang, Y.; Wang, J.; Li, Q. Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Sci. 2009, 18, 936–948. [Google Scholar] [CrossRef] [PubMed]
  46. Munasinghe, T.S.; Edwards, M.R.; Tsimbalyuk, S.; Vogel, O.A.; Smith, K.M.; Stewart, M.; Foster, J.K.; Bosence, L.A.; Aragão, D.; Roby, J.A.; et al. MERS-CoV ORF4b employs an unusual binding mechanism to target IMPα and block innate immunity. Nat. Commun. 2022, 13, 1604. [Google Scholar] [CrossRef] [PubMed]
  47. Studier, F.W. Protein production by auto-induction in high-density shaking cultures. Protein Expr. Purif. 2005, 41, 207–234. [Google Scholar] [CrossRef] [PubMed]
  48. Hyberts, S.G.; Takeuchi, K.; Wagner, G. Poisson-gap sampling and forward maximum entropy reconstruction for enhancing the resolution and sensitivity of protein NMR data. J. Am. Chem. Soc. 2010, 132, 2145–2147. [Google Scholar] [CrossRef]
  49. Kazimierczuk, K.; Orekhov, V.Y. Accelerated NMR spectroscopy by using compressed sensing. Angew. Chem. Int. Ed. 2011, 50, 5556–5559. [Google Scholar] [CrossRef]
  50. Delaglio, F.; Grzesiek, S.; Vuister, G.W.; Zhu, G.; Pfeifer, J.; Bax, A. NMRPipe: A multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 1995, 6, 277–293. [Google Scholar] [CrossRef]
  51. Lee, W.; Tonelli, M.; Markley, J.L. NMRFAM-SPARKY: Enhanced software for biomolecular NMR spectroscopy. Bioinformatics 2015, 31, 1325–1327. [Google Scholar] [CrossRef] [PubMed]
  52. Ayed, A.; Mulder, F.A.; Yi, G.-S.; Lu, Y.; Kay, L.E.; Arrowsmith, C.H. Latent and active p53 are identical in conformation. Nat. Struct. Mol. Biol. 2001, 8, 756–760. [Google Scholar] [CrossRef] [PubMed]
  53. Battye, T.G.G.; Kontogiannis, L.; Johnson, O.; Powell, H.R.; Leslie, A.G. iMOSFLM: A new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr. Sect. D Biol. Crystallogr. 2011, 67, 271–281. [Google Scholar] [CrossRef]
  54. Winn, M.D.; Ballard, C.C.; Cowtan, K.D.; Dodson, E.J.; Emsley, P.; Evans, P.R.; Keegan, R.M.; Krissinel, E.B.; Leslie, A.G.; McCoy, A.; et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. Sect. D Biol. Crystallogr. 2011, 67, 235–242. [Google Scholar] [CrossRef]
  55. McCoy, A.J.; Grosse-Kunstleve, R.W.; Adams, P.D.; Winn, M.D.; Storoni, L.C.; Read, R.J. Phaser crystallographic software. J. Appl. Crystallogr. 2007, 40, 658–674. [Google Scholar] [CrossRef] [PubMed]
  56. Liebschner, D.; Afonine, P.V.; Baker, M.L.; Bunkóczi, G.; Chen, V.B.; Croll, T.I.; Hintze, B.; Hung, L.-W.; Jain, S.; McCoy, A.J.; et al. Macromolecular structure determination using X-rays, neutrons and electrons: Recent developments in Phenix. Acta Crystallogr. Sect. D Biol. Crystallogr. 2019, 75, 861–877. [Google Scholar] [CrossRef]
  57. Vagin, A.A.; Steiner, R.A.; Lebedev, A.A.; Potterton, L.; McNicholas, S.; Long, F.; Murshudov, G.N. REFMAC5 dictionary: Organization of prior chemical knowledge and guidelines for its use. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004, 60, 2184–2195. [Google Scholar] [CrossRef] [PubMed]
  58. Palasingam, P.; Jauch, R.; Ng, C.K.L.; Kolatkar, P.R. The structure of Sox17 bound to DNA reveals a conserved bending topology but selective protein interaction platforms. J. Mol. Biol. 2009, 388, 619–630. [Google Scholar] [CrossRef]
  59. Jauch, R.; Ng, C.K.; Narasimhan, K.; Kolatkar, P.R. The crystal structure of the Sox4 HMG domain–DNA complex suggests a mechanism for positional interdependence in DNA recognition. Biochem. J. 2012, 443, 39–47. [Google Scholar] [CrossRef]
  60. Dodonova, S.O.; Zhu, F.; Dienemann, C.; Taipale, J.; Cramer, P. Nucleosome-bound SOX2 and SOX11 structures elucidate pioneer factor function. Nature 2020, 580, 669–672. [Google Scholar] [CrossRef]
  61. Williams, D.C., Jr.; Cai, M.; Clore, G.M. Molecular basis for synergistic transcriptional activation by Oct1 and Sox2 revealed from the solution structure of the 42-kDa Oct1· Sox2· Hoxb1-DNA ternary transcription factor complex. J. Biol. Chem. 2004, 279, 1449–1457. [Google Scholar] [CrossRef] [PubMed]
  62. Murphy, E.C.; Zhurkin, V.B.; Louis, J.M.; Cornilescu, G.; Clore, G.M. Structural basis for SRY-dependent 46-X, Y sex reversal: Modulation of DNA bending by a naturally occurring point mutation. J. Mol. Biol. 2001, 312, 481–499. [Google Scholar] [CrossRef] [PubMed]
  63. Klaus, M.; Prokoph, N.; Girbig, M.; Wang, X.; Huang, Y.-H.; Srivastava, Y.; Hou, L.; Narasimhan, K.; Kolatkar, P.R.; Francois, M.; et al. Structure and decoy-mediated inhibition of the SOX18/Prox1-DNA interaction. Nucleic Acids Res. 2016, 44, 3922–3935. [Google Scholar] [CrossRef] [PubMed]
  64. Joint Center for Structural Genomics (JCSG); Partnership for Stem Cell Biology (STEMCELL). Crystal Structure of a HMG Domain of Transcription Factor SOX-9 Bound to DNA (SOX-9/DNA) from Homo Sapiens at 2.77 Å Resolution. Available online: https://www.rcsb.org/structure/4EUW (accessed on 31 May 2024).
  65. Gao, N.; Jiang, W.; Gao, H.; Cheng, Z.; Qian, H.; Si, S.; Xie, Y. Structural basis of human transcription factor Sry-related box 17 binding to DNA. Protein Pept. Lett. 2013, 20, 481–488. [Google Scholar] [PubMed]
  66. Cary, P.D.; Read, C.M.; Davis, B.; Driscoll, P.C.; Crane-Robinson, C. Solution structure and backbone dynamics of the DNA-binding domain of mouse Sox-5. Protein Sci. 2001, 10, 83–98. [Google Scholar] [CrossRef]
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Figure 1. SOX family members share a similar HMG-box domain. Amino acid sequence alignment of the HMG-box domains from human SOX family members, coloured by group and numbered as per full-length UniProt sequences; conserved residues are shown in bold. A conservation bar is shown at the top of the alignment in grey, numbered as per HMG-box numbering; upper case = conserved residue, lower case = most common residue. The three α-helices of the HMG-box domain are indicated at the bottom of the alignment. The black dashed box highlights the region proposed to be critical for RNA binding. The alignment was produced using UGENE software version 36.0 [15].
Figure 1. SOX family members share a similar HMG-box domain. Amino acid sequence alignment of the HMG-box domains from human SOX family members, coloured by group and numbered as per full-length UniProt sequences; conserved residues are shown in bold. A conservation bar is shown at the top of the alignment in grey, numbered as per HMG-box numbering; upper case = conserved residue, lower case = most common residue. The three α-helices of the HMG-box domain are indicated at the bottom of the alignment. The black dashed box highlights the region proposed to be critical for RNA binding. The alignment was produced using UGENE software version 36.0 [15].
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Figure 2. Comparison between purification profiles of SOX17 and SOX30 HMG-box domains. (A,B) SOX17 (Group F) and SOX30 (Group H) HMG-box domains were first purified via affinity chromatography (left panel). Following affinity purification, proteins were either left untreated or treated with DNase or RNase, before further purification via analytical gel filtration (right panel). Gel samples were taken of whole cell (WC), supernatant (SN), flowthrough (FT), purified eluant (P), purified eluant treated with DNase (PD), and purified eluant treated with RNase (PR) and analysed on agarose gels, for visualisation of nucleic acids, and via SDS-PAGE, for visualisation of protein. L = ladder. (A) The SOX17 HMG-box domain co-purifies with RNA during affinity and analytical gel filtration chromatography. In no treatment and DNase-treated samples, SOX17 HMG-box elutes around 17 to 19 mL, and RNA can be detected in fractions 10 to 19. In RNase-treated samples, the RNA-related peak shifts to fraction 22. As visualised on the agarose gels, the majority of the nucleic acids were attributed to RNA, with the least seen following RNase treatment. (B) The SOX30 HMG-box domain does not co-purify with RNA. In no treatment, DNase-treated, and RNase-treated samples, SOX30 HMG-box elutes around 17 to 19 mL, with no RNA detected in any of the fractions. Further, no nucleic acids are detected on the agarose gels.
Figure 2. Comparison between purification profiles of SOX17 and SOX30 HMG-box domains. (A,B) SOX17 (Group F) and SOX30 (Group H) HMG-box domains were first purified via affinity chromatography (left panel). Following affinity purification, proteins were either left untreated or treated with DNase or RNase, before further purification via analytical gel filtration (right panel). Gel samples were taken of whole cell (WC), supernatant (SN), flowthrough (FT), purified eluant (P), purified eluant treated with DNase (PD), and purified eluant treated with RNase (PR) and analysed on agarose gels, for visualisation of nucleic acids, and via SDS-PAGE, for visualisation of protein. L = ladder. (A) The SOX17 HMG-box domain co-purifies with RNA during affinity and analytical gel filtration chromatography. In no treatment and DNase-treated samples, SOX17 HMG-box elutes around 17 to 19 mL, and RNA can be detected in fractions 10 to 19. In RNase-treated samples, the RNA-related peak shifts to fraction 22. As visualised on the agarose gels, the majority of the nucleic acids were attributed to RNA, with the least seen following RNase treatment. (B) The SOX30 HMG-box domain does not co-purify with RNA. In no treatment, DNase-treated, and RNase-treated samples, SOX30 HMG-box elutes around 17 to 19 mL, with no RNA detected in any of the fractions. Further, no nucleic acids are detected on the agarose gels.
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Figure 3. (A) EMSA results show that all SOX HMG-box domains tested, except for SOX30, alter the migration of a 60-mer ssDNA nucleic acid probe, indicating direct binding. Proteins were stained with Coomassie blue (top panel; green), the nucleic acid probe was stained with GelRed (middle panel; red), and the overlay is displayed in the bottom panel, with the complexes shown in dark green. Arrows indicate sample loading position; F indicates free 60-mer nucleic acid probe. (B) Fluorescence polarisation assays measuring binding affinity between SOX proteins and a FAM-labelled RNA probe verified the EMSA results. SOX17 (green) bound RNA with a Kd of ~327 nM, with no RNA binding detected for SOX30 (red). SOX2 (blue) was run as a positive control and bound RNA with a Kd of ~57 nM. Data shown as n = 3; error bars represent mean ± standard error of the mean; ND = not determined.
Figure 3. (A) EMSA results show that all SOX HMG-box domains tested, except for SOX30, alter the migration of a 60-mer ssDNA nucleic acid probe, indicating direct binding. Proteins were stained with Coomassie blue (top panel; green), the nucleic acid probe was stained with GelRed (middle panel; red), and the overlay is displayed in the bottom panel, with the complexes shown in dark green. Arrows indicate sample loading position; F indicates free 60-mer nucleic acid probe. (B) Fluorescence polarisation assays measuring binding affinity between SOX proteins and a FAM-labelled RNA probe verified the EMSA results. SOX17 (green) bound RNA with a Kd of ~327 nM, with no RNA binding detected for SOX30 (red). SOX2 (blue) was run as a positive control and bound RNA with a Kd of ~57 nM. Data shown as n = 3; error bars represent mean ± standard error of the mean; ND = not determined.
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Figure 4. 15N,1H HSQC-monitored NMR titration of 15N-labelled SOX17 HMG-box domain indicates the importance of the C-terminal residues for binding a 12-mer ssDNA nucleic acid probe. (A) Plot of the change in average 1HN and 15N chemical shifts (blue indicates residues with 1 standard deviation (SD) of the mean of chemical shift; red indicates residues with 2 SD). More shift in a residue means more conformational change in the interaction with the nucleic acid probe. (B) The crystal structure of the SOX17 HMG-box domain bound to DNA (PDB ID: 3F27) [58], highlighting the position of residues with significant chemical shifts (blue indicates residues with 1 SD of the mean of chemical shift; red indicates residues with 2 SD). (C) 1H,15N HSQC spectrum indicating chemical shift dependence on the presence of a 12-mer ssDNA nucleic acid probe. Red (no ssDNA), yellow (25 µM ssDNA), orange (50 µM ssDNA), and cyan (100 µM ssDNA).
Figure 4. 15N,1H HSQC-monitored NMR titration of 15N-labelled SOX17 HMG-box domain indicates the importance of the C-terminal residues for binding a 12-mer ssDNA nucleic acid probe. (A) Plot of the change in average 1HN and 15N chemical shifts (blue indicates residues with 1 standard deviation (SD) of the mean of chemical shift; red indicates residues with 2 SD). More shift in a residue means more conformational change in the interaction with the nucleic acid probe. (B) The crystal structure of the SOX17 HMG-box domain bound to DNA (PDB ID: 3F27) [58], highlighting the position of residues with significant chemical shifts (blue indicates residues with 1 SD of the mean of chemical shift; red indicates residues with 2 SD). (C) 1H,15N HSQC spectrum indicating chemical shift dependence on the presence of a 12-mer ssDNA nucleic acid probe. Red (no ssDNA), yellow (25 µM ssDNA), orange (50 µM ssDNA), and cyan (100 µM ssDNA).
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Figure 5. The C-terminal region of the SOX17 HMG-box domain is critical for RNA binding. (A) Analytical gel filtration profiles of SOX17 wild-type (SOX17 WT), N-terminal truncation (SOX17 ∆N), C-terminal truncation (SOX17 ∆C), and N- and C-terminal truncation (SOX17 ∆CN) HMG-box domain constructs. Co-purification of RNA is observed with SOX17 WT and SOX17 ∆N, while no RNA co-purification is evident with SOX17 ∆C or SOX17 ∆CN. (B) EMSA between WT and truncated SOX17 constructs and a 22-mer ssDNA nucleic acid probe; SOX30 was used as a negative control. SOX17 WT and SOX17 ∆N can bind to the nucleic acid probe and shift its position, while C-terminal truncation of the HMG-box domain abolishes RNA binding, as evident in SOX17 ∆C and SOX17 ∆CN. Arrows indicate sample loading position; F indicates free 22-mer nucleic acid probe. (C) Fluorescence polarisation assays measuring binding affinity between WT and truncated SOX17 proteins and a FAM-labelled RNA probe. SOX17 WT (green) bound RNA with a Kd of ~327 nM, with no RNA binding detected for SOX17 ∆C (orange) or SOX17 ∆CN (purple). Some RNA binding was detected at the highest concentrations of SOX17 ∆N (pink); however, the binding affinity was too low to be determined. SOX17 WT data as shown in Figure 3B. Data shown as n = 3; error bars represent mean ± standard error of the mean; ND = not determined. (D) Models of the SOX17 HMG-box domain in SOX17 WT, SOX17 ∆N, SOX17 ∆C, and SOX17 ∆CN constructs. The N-terminal truncated region is shown in red; the C-terminal truncated region is shown in blue. (E) Sequences of the WT and truncated SOX17 HMG-box domain constructs used, numbered as per full-length UniProt sequences.
Figure 5. The C-terminal region of the SOX17 HMG-box domain is critical for RNA binding. (A) Analytical gel filtration profiles of SOX17 wild-type (SOX17 WT), N-terminal truncation (SOX17 ∆N), C-terminal truncation (SOX17 ∆C), and N- and C-terminal truncation (SOX17 ∆CN) HMG-box domain constructs. Co-purification of RNA is observed with SOX17 WT and SOX17 ∆N, while no RNA co-purification is evident with SOX17 ∆C or SOX17 ∆CN. (B) EMSA between WT and truncated SOX17 constructs and a 22-mer ssDNA nucleic acid probe; SOX30 was used as a negative control. SOX17 WT and SOX17 ∆N can bind to the nucleic acid probe and shift its position, while C-terminal truncation of the HMG-box domain abolishes RNA binding, as evident in SOX17 ∆C and SOX17 ∆CN. Arrows indicate sample loading position; F indicates free 22-mer nucleic acid probe. (C) Fluorescence polarisation assays measuring binding affinity between WT and truncated SOX17 proteins and a FAM-labelled RNA probe. SOX17 WT (green) bound RNA with a Kd of ~327 nM, with no RNA binding detected for SOX17 ∆C (orange) or SOX17 ∆CN (purple). Some RNA binding was detected at the highest concentrations of SOX17 ∆N (pink); however, the binding affinity was too low to be determined. SOX17 WT data as shown in Figure 3B. Data shown as n = 3; error bars represent mean ± standard error of the mean; ND = not determined. (D) Models of the SOX17 HMG-box domain in SOX17 WT, SOX17 ∆N, SOX17 ∆C, and SOX17 ∆CN constructs. The N-terminal truncated region is shown in red; the C-terminal truncated region is shown in blue. (E) Sequences of the WT and truncated SOX17 HMG-box domain constructs used, numbered as per full-length UniProt sequences.
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Figure 6. The C-terminal region of the HMG-box domains of SRY, SOX2, and SOX11 similarly play an important role in RNA binding. (A) Aligned amino acid sequences of SRY, SOX2, and SOX11 HMG-box domains, numbered as per full-length UniProt sequences. Conserved residues are shown in bold. Residues removed in C-terminal truncation (∆C) constructs, proposed to be critical for RNA binding, are shown in red. (B–D) SEC graphs of HMG-box domain wild-type (WT) and ∆C constructs of SRY (B), SOX2 (C), and SOX11 (D). SRY WT, SOX2 WT, and SOX11 WT all co-purify with RNA, while SRY ∆C, SOX2 ∆C, and SOX11 ∆C do not, demonstrating that C-terminal truncation of the HMG-box domain disrupts RNA binding.
Figure 6. The C-terminal region of the HMG-box domains of SRY, SOX2, and SOX11 similarly play an important role in RNA binding. (A) Aligned amino acid sequences of SRY, SOX2, and SOX11 HMG-box domains, numbered as per full-length UniProt sequences. Conserved residues are shown in bold. Residues removed in C-terminal truncation (∆C) constructs, proposed to be critical for RNA binding, are shown in red. (B–D) SEC graphs of HMG-box domain wild-type (WT) and ∆C constructs of SRY (B), SOX2 (C), and SOX11 (D). SRY WT, SOX2 WT, and SOX11 WT all co-purify with RNA, while SRY ∆C, SOX2 ∆C, and SOX11 ∆C do not, demonstrating that C-terminal truncation of the HMG-box domain disrupts RNA binding.
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Figure 7. Crystal structure of the SOX30 HMG-box domain and comparison with other SOX family member HMG-box domain structures. (A) Structure of the SOX30 HMG-box domain (residues 335–405) at 0° and 90°, showing the typical three α-helices arranged in an L-shape and flanked by two disordered regions at the N- and C-termini. (B) Superimposition of the SOX30 HMG-box domain with other SOX protein HMG-box domains, demonstrating a very similar secondary structure between SOX family members. The seven amino acids identified as critical for RNA binding (73–79, HMG-box numbering) are located in the disordered C-terminal end of the HMG-box domain and are thus not visible in the SOX30 structure or the structures of Sox5, SOX9, and SOX17. SOX30 is shown in cyan; Sox4 is shown in dark orange (PDB ID: 3U2B); Sox18 is shown in light green (PDB ID: 4Y60); SOX11 is shown in light orange (PDB ID: 6T78); SOX2 is shown in magenta (PDB ID: 1O4X); SRY is shown in blue (PDB ID: 1J46); SOX17 is shown in green (PDB ID: 4A3N); SOX9 is shown in yellow (PDB ID: 4EUW); Sox5 is shown in grey (PDB ID: 1I11).
Figure 7. Crystal structure of the SOX30 HMG-box domain and comparison with other SOX family member HMG-box domain structures. (A) Structure of the SOX30 HMG-box domain (residues 335–405) at 0° and 90°, showing the typical three α-helices arranged in an L-shape and flanked by two disordered regions at the N- and C-termini. (B) Superimposition of the SOX30 HMG-box domain with other SOX protein HMG-box domains, demonstrating a very similar secondary structure between SOX family members. The seven amino acids identified as critical for RNA binding (73–79, HMG-box numbering) are located in the disordered C-terminal end of the HMG-box domain and are thus not visible in the SOX30 structure or the structures of Sox5, SOX9, and SOX17. SOX30 is shown in cyan; Sox4 is shown in dark orange (PDB ID: 3U2B); Sox18 is shown in light green (PDB ID: 4Y60); SOX11 is shown in light orange (PDB ID: 6T78); SOX2 is shown in magenta (PDB ID: 1O4X); SRY is shown in blue (PDB ID: 1J46); SOX17 is shown in green (PDB ID: 4A3N); SOX9 is shown in yellow (PDB ID: 4EUW); Sox5 is shown in grey (PDB ID: 1I11).
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Table 1. Data collection and refinement statistics. Statistics for the highest resolution shell are shown in parentheses.
Table 1. Data collection and refinement statistics. Statistics for the highest resolution shell are shown in parentheses.
Data Collection and ProcessingSOX30 HMG-Box
  Wavelength (Å)0.95372
  Resolution range (Å)17.99–1.4 (1.4–1.42)
  Space groupP212121
  Unit cell (Å, °)33.7, 35.7, 52.91, 90, 90, 90
  Unique reflections13155 (656)
  Multiplicity11.8 (9.9)
  Completeness (%)99.9 (99.0)
  Mean I/sigma(I)17.8 (4.4)
  Wilson B-factor Å211.04
  Rpim0.029 (0.19)
  CC(1/2)0.999 (0.968)
Refinement
  Number of reflections13111
  Number of R-free reflections626
  R-work (%)17.37
  R-free (%)19.92
  RMS(bonds)0.0093
  RMS(angles)2.583
  Ramachandran plot
    favoured (%)100
    allowed (%)0
    outliers (%)0
PDB accession code7JJK
Table 2. SOX protein HMG-box domain structures and their similarity to the SOX30 HMG-box domain structure. RMSD calculated for the region encompassing helices α1 to α3.
Table 2. SOX protein HMG-box domain structures and their similarity to the SOX30 HMG-box domain structure. RMSD calculated for the region encompassing helices α1 to α3.
ProteinPDB ID (Reference)Resolution (Å)RMSD to SOX30 (Å)
Sox43U2B [59]2.400.613 [over 47 Cα]
SOX116T78 [60]2.500.676 [over 47 Cα]
SOX21O4X [61]NMR0.726 [over 49 Cα]
SRY1J46 [62]NMR0.755 [over 52 Cα]
Sox184Y60 [63]1.750.766 [over 54 Cα]
SOX94EUW [64]2.770.918 [over 52 Cα]
SOX174A3N [65]2.400.925 [over 53 Cα]
Sox51I11 [66]NMR1.350 [over 49 Cα]
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MDPI and ACS Style

Ghafoori, S.M.; Sethi, A.; Petersen, G.F.; Tanipour, M.H.; Gooley, P.R.; Forwood, J.K. RNA Binding Properties of SOX Family Members. Cells 2024, 13, 1202. https://doi.org/10.3390/cells13141202

AMA Style

Ghafoori SM, Sethi A, Petersen GF, Tanipour MH, Gooley PR, Forwood JK. RNA Binding Properties of SOX Family Members. Cells. 2024; 13(14):1202. https://doi.org/10.3390/cells13141202

Chicago/Turabian Style

Ghafoori, Seyed Mohammad, Ashish Sethi, Gayle F. Petersen, Mohammad Hossein Tanipour, Paul R. Gooley, and Jade K. Forwood. 2024. "RNA Binding Properties of SOX Family Members" Cells 13, no. 14: 1202. https://doi.org/10.3390/cells13141202

APA Style

Ghafoori, S. M., Sethi, A., Petersen, G. F., Tanipour, M. H., Gooley, P. R., & Forwood, J. K. (2024). RNA Binding Properties of SOX Family Members. Cells, 13(14), 1202. https://doi.org/10.3390/cells13141202

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