COBALT was used to perform multiple sequence alignment to identify conserved regions among SERBP1 homologues from eleven different species (nine different phyla) (Papadopoulos and Agarwala, 2007). Amino-acid identity was prioritized, and an amino-acid was defined as conserved if it was present in at least seven species. Protein sequences were obtained from the NCBI (https://www.ncbi.nlm.nih.gov/) with the following accession numbers: H. sapiens (NP_001018077.1), Saccoglossus kowalevskii (XP_002740974.1), Acanthaster planci (XP_022107686.1), Drosophila melanogaster (NP_523572.1), Stegodyphus mimosarum (KFM69747.1), Centruroides sculpturatus (XP_023216060.1), Octopus sinensis (XP_029640355.1), Teladorsagia circumcincta (PIO76243.1), Echinococcus granulosus (XP_024349465.1), Nematostella vectensis (XP_001637231.1), A. queenslandica (XP_011406826.2).

The SERBP1 189–400 construct (Addgene accession number 172315) was expressed in E.coli as previously described (Baudin et al., 2021). The genes for full-length SERBP1 and a SERBP1 149-400 construct were amplified by PCR (primer are shown in Supplementary Table S1), digested by KasI and BamHI restriction enzymes and cloned into a custom pAG8Ha-His vector that introduced an 8x histidine tag followed by a TEV cleavage site N-terminal to the coding sequence. Plasmids were transformed by heat shock into E. coli BL21 Star™ (DE3) (Invitrogen, MA) and a colony from the resultant agar plate was used to inoculate a 5 ml LB starter culture that was grown at 37^°C for 6–8 h. The LB starter culture was used to inoculate a 100 ml M9 preculture that was grown overnight at 37^°C with shaking. The overnight preculture was then used to inoculate an expression culture of 900 ml M9 minimal media. Both the 100 ml starter and 900 ml expression cultures were supplemented with ¹⁵NH₄Cl (¹³C-glucose) for isotopic enrichment. For non-isotopically enriched protein, the overnight culture was 10 ml LB and 4 ml was used to inoculate a 1 L LB expression culture. All cultures were supplemented with 100 μg/ ml of ampicillin. Expression cultures were grown at 37°C in baffled Fernbach flasks with shaking, and protein expression was induced at OD₆₀₀ ∼0.6–0.8 with 1 mM IPTG and continued for 3 or 6 h, for LB or M9 cultures, respectively. The cells were harvested by centrifugation at 4000 g for 20 min and the resulting pellets were stored at −80°C.

SERBP1 constructs were purified as previously described for SERBP1 189–400 (Baudin et al., 2021). Briefly, frozen E. coli pellets were thawed and resuspended in 8 M urea, 50 mM Tris pH 8.0, 150 mM NaCl, 20 mM imidazole, lysed by sonication (6 cycles of 10 s on, 30 s off), and the lysate was cleared by centrifugation for 30 min at 45000 g at 4°C. The supernatant was applied to a 5 ml HisTrap HP column (Cytiva, MA, United States), equilibrated with the lysis buffer, washed with 25 column volumes of the same buffer, and eluted with 8 M urea, 50 mM Tris pH 8.0, 150 mM NaCl, 500 mM imidazole. Eluted protein was concentrated with a 3 kDa (149–400, 189–400) or 10 kDa (full-length) cutoff Amicon centrifugal concentrator (Merck, NJ, United States) to ∼4 ml and diluted into 40 ml of 100 mM sodium phosphate buffer, pH 7, 1 mM PMSF, 1 mM EDTA buffer. Precipitated protein was removed by centrifugation at 3,000 g for 15 min, and the protein was then dialyzed against 100 mM sodium phosphate buffer pH 7 (3 x 2 L), on the third change 500 µL of 1.6 mg/ ml Tobacco Etch Virus (TEV) protease was added and the mixture was incubated at room temperature overnight. The 8x His tag was not removed from full-length SERBP1. Post-dialysis, the sample was centrifugated at 3,000 g for 15 min to remove any precipitate and concentrated to approximately 4 ml with an Amicon centrifugal concentrator. A final cation exchange chromatography polishing step was used for SERBP1 149–400 and 189–400. The 4 ml sample was diluted into 60 ml with 50 mM sodium acetate buffer pH 4.5, 0.5 mM PMSF, 0.5 mM EDTA, loaded on a 5 ml SP Sepharose fast-flow column (Cytiva, MA, United States), washed with 20 column volumes of the same buffer and eluted with a 0.05–1 M NaCl gradient. Fractions containing SERBP 149–400 were concentrated, and buffer exchanged into NMR buffer (20 mM sodium phosphate buffer pH 6.9, 60 mM NaCl, 1 mM PMSF, 0.2 mM EDTA, 10 % D₂O).

All experiments were recorded on a Bruker Avance NEO spectrometer operating at a proton Larmor frequency of 700.13 MHz, at a temperature of 5°C using a 5 mm TCI z-axis gradient cryogenic probe. Data were processed with the NMRPipe software suite (Delaglio et al., 1995) and analyzed with CCPNMR Analysis 2.5 software (Skinner et al., 2016). SERBP1 149–400 ¹H, ¹³C_α, ¹³C_β, ¹³C’ and ¹⁵N backbone resonances were assigned through the analysis of a set of 2D and 3D experiments, namely ¹H,¹⁵N-HSQC, HNCACB, CBCA(CO)NH, HNCO, HN(CA)CO and HCC(CO)NH, recorded on a ¹³C,¹⁵N-labeled sample at a concentration of 300 μM, in NMR buffer. The ¹H,¹⁵N-HSQC was recorded with 128*x 1024* complex points in the indirect (¹⁵N) and direct (¹H) dimensions, corresponding to acquisition times of 75.2 and 112.6 ms, respectively. Acquisition parameters for the HNCO and HN(CA)CO consisted of 32^∗ x 64^∗ x 1024^∗ complex points in the indirect (F1, ¹³C), (F2, ¹⁵N) and direct (F3 ¹H) dimensions, corresponding to acquisition times of 16.5, 37.6, 112.6 ms, respectively; acquisition parameters for the HNCACB, CBCA(CO)NH and HCC(CO)NH consisted of 128^∗ x 64^∗ x 1024^∗ complex points in the indirect (F1, ¹³C), (F2, ¹⁵N) and direct (F3 ¹H) dimensions, corresponding to acquisition times of 11, 37.6, 112.6 ms, respectively. All 3D experiments were recorded in non-uniform sampling (NUS) mode (Delaglio et al., 2017) with a sampling density of 20%, and the spectra were reconstructed using the SMILE algorithm implemented in NMRPipe (Ying et al., 2017).

¹⁵N R ₁ and R ₂ relaxation rates were calculated from T ₁ and T _1ρ experiments, recorded on 50 and 100 µM samples for SERBP1 189–400 and SERBP1 149–400, respectively, in NMR buffer using 64* x 1024* complex data points in the indirect (¹⁵N) and direct (¹H) dimensions corresponding to acquisition times of 37.6 and 112.6 ms, respectively. The ¹⁵N T ₁ experiment consisted of eight interleaved spectra with the following relaxation delays: 40, 80, 200, 280, 300, 400, 600, and 800 ms. The T _1ρ experiment was recorded using a B ₁ field of 1400 Hz and eight interleaved spectra with the following relaxation delays: 1, 21, 31, 41, 61, 81, 121 and 161 ms. ¹⁵N R ₂ rates were calculated using the following equation (Massi et al., 2004): R 1 ρ = R 1 cos 2 θ + R 2 sin 2 θ   (1) with θ = arctan(ω ₁/Ω), where ω ₁ is the B ₁ field strength (here 1400 Hz) and Ω is the offset from the spinlock carrier frequency. ¹H-¹⁵N heteronuclear NOE experiments were recorded on the same samples and consisted of two interleaved experiments, with and without proton saturation, using a recycle delay of 4 s. Spectra were acquired with 64^∗ x 1024^∗ complex data points in the indirect (¹⁵N) and direct (¹H) dimensions corresponding to acquisition times of 37.6 and 112.6 ms, respectively.

For RNA binding experiments, SERBP1 189–400 or 149–400 samples were diluted to 50 µM in 500 µL in NMR buffer and added to a 5 mm NMR tube. The RNA sequence 5′-GCGCGGG-3′, representing a G-quartet, was synthesized (IDT, IA), desalted, dried, and resuspended in RNAase-free water (Qiagen, MD) to a concentration of 3.125 mM. The RNA stock was titrated into the SERBP1 sample to final RNA:SERBP1 ratios of 2:5, 4:5, 6:5, and 8:5. This required the addition of a maximum of 12 µL of the RNA stock, thus the effect of dilution is negligible. ¹H,¹⁵N-HSQC spectra were recorded at 4°C for each titration point with 64^∗ x 1024^∗ complex data points in the indirect (¹⁵N) and direct (¹H) dimensions corresponding to acquisition times of 37.6 and 112.6 ms, respectively. Spectra were apodized with a sine bell function and zero filled to twice the number of acquired points for data analysis. Chemical shift perturbations (CSP) were calculated by weighting the ¹H and ¹⁵N chemical shifts with respect to their gyromagnetic ratio using the following equation (Williamson, 2013): Δδ = ( δ   1 H ) 2 + 0.1 ( δ 15 N ) 2 (2) CSPs were considered significant when they were higher than the standard deviation of Δδ_max for all residues (Williamson, 2013).

Circular dichroic spectra were recorded on 10 µM samples of SERBP1 149–400 or 189–400 dissolved in 20 mM Na₂HPO₄ pH 6.9, 60 mM NaCl in a 2 mm pathlength circular cuvette using a Jasco 810 spectropolarimeter (Jasco, OK) at a scan speed of 50 nm/ min with 0.5 nm. Temperature was controlled with a recirculating external water bath and was allowed to equilibrate for 20 min before recording data after each ramp. Each temperature point (20–80°C in 10°C steps) was recorded in triplicate, averaged and converted to mean residual ellipticity using previously described relationships (Chemes et al., 2012).

Size exclusion chromatography (SEC) experiments were performed using a BioRad NGC fast-performance liquid chromatography system (BioRad, CA, United States) equipped with a Superdex 200 10 x 300 mm analytical size exclusion column (Cytiva, MA, United States), equilibrated with NMR buffer (20 mM Na₂HPO₄ pH 6.9, 60 mM NaCl, 1 mM PMSF, 0.2 mM EDTA) in the presence or absence of 2 M guanidinium hydrochloride (GdnHCl). 200 µL of a 15 µM sample of SERBP1 189–400 or 149–400 was applied to the column. The flow rate was 0.5 ml/ min, and elution was monitored at three wavelengths: 215, 280, and 340 nm.

SERBP1 189–400 and 149–400 stock samples were diluted to a concentration of 25 µM into 20 mM Na₂HPO₄ pH 7.0, 60 mM NaCl buffer, in presence or absence of 2 M GdnHCl. 360 µL samples were loaded into one sector of a 12 mm double-sector epon-filled centerpiece, and 400 µL of a reference solution of the sample buffer was loaded into the other sector. Radial absorbance scans were collected at 4 min intervals at 280 nm, using an XL-1 analytical centrifuge (Beckman Coulter, CA, United States) equipped with a Ti60 rotor at 40000 rpm, 20°C. Total experiment time was ∼10 h. Data was fit using a continuous c(s) distribution model using the SEDFIT software (Schuck, 2000), using adjusted buffer density (1.04861) and buffer relative viscosity (1.09833) values to account for GdnHCl.

A SERBP1 189–400 stock sample was diluted to a concentration of 125 µM in 60 µL of 20 mM Na₂HPO₄ pH 7.0, 60 mM NaCl buffer, in presence or absence of 2 M GdnHCl, and transferred to a low-volume quartz cuvette. Measurements were conducted on a DynaPro NanoStar dynamic light scattering (DLS) instrument (Wyatt, CA, United States) at 20°C. For each sample 150 10 s acquisitions were collected and averaged. Data were analyzed using the algorithms available in the instrument control software and output as size distributions. Experimentally measured R _h values were compared with calculated R _h values using the following equation proposed by Marsh and Forman-Kay to account for intrinsic disorder (Marsh and Forman-Kay, 2010): R h IDP = 2.49 N 0.509 (3) where N is the number of residues in the protein polymer chain.

SERBP1 149–400 was labeled with the DyLight™ 650 fluorophore (Thermo Fisher Scientific, MO, United States) at the N-terminus using S. aureus sortase A following established protocols (Theile et al., 2013; Antos et al., 2017). The sortase-recognition peptide KLPETGG was synthesized, HPLC purified, lyophilized (Genscript, NJ) and reacted with the fluorophore following the manufacturer’s instructions. The fluorescently labeled peptide and SERBP1 were mixed at a 1:1 molar ratio. Recombinant sortase A was added to a final concentration of 2.5 µM and the reaction was allowed to proceed overnight at room temperature. Labeled SERBP1 was separated from unconjugated peptide and free fluorophore by passing over a Superdex 75 HiLoad 16/60 size exclusion column (Cytiva, MA, United States).

Torula yeast RNA (MilliporeSigma, MA, United States) was dissolved at 10 mg/ml in 20 mM Tris pH 7.4, 150 mM NaCl, centrifuged at 13000 g for 10 min, desalted using a PD-10 desalting column (Cytiva, MA, United States), and diluted to a final stock concentration of 3.6 mg/ml determined by A₂₆₀. Unlabeled SERBP1 149–400 was mixed with DyLight 650-labeled SERBP1 149–400 at a molar ratio of 0.03% and dissolved to 6 or 12 µM in 154 mM NaCl, 64 mM Tris pH 7.5, 12.8 % (v/v) glycerol, 1.28 mM DTT, 12.8 % (w/v) PEG3000. Stock RNA was added to a final concentration of 0.4, 0.2, or 0.05 mg/ ml. Samples of SERBP1 189–400 were prepared identically except without fluorescent labeling. Control samples consisting of buffer only or buffer plus RNA only remained clear. SERBP1 samples used in the salt series were prepared similarly except the buffer the protein was dissolved in contained only 20 mM Tris pH 7.5 and 0, 0.05, 0.15, 0.3, or 1 M NaCl.

Phase separation was assayed by transferring 4.5 µL samples to chambered glass coverslips (Grace Biolabs, OR). Chambers were sealed with a second coverslip to reduce evaporation and incubated for 10 min at ambient temperature before imaging with an Olympus FV3000 inverted confocal microscope (Olympus, PA, United States) operating at 1% laser power on the 640 nm channel. Images were acquired simultaneously in differential interference contrast (DIC) and fluorescent modes. Fluorescence recovery after photobleaching (FRAP) measurements were conducted on 12 µM SERBP1 149–400 samples with low (0.05 mg/ ml) or no RNA, and generally recovered to ∼80% of the initial fluorescent intensity. Image contrast was adjusted globally, and droplet area was measured using the appropriate subroutines in Fiji (ImageJ) (Schindelin et al., 2012).

The largest and predominant SERBP1 isoform contains 408 amino-acids encompassing distinct domains including two hyaluronic acid binding protein (HABP) homology domains, IHABP4 (intracellular HABP4, spanning residues 5–152) and HABP4 (residues 189–314) as indicated by UniProtKB entry Q8NC51 (Figure 1A). Two distinct RG/RGG repeats or boxes comprising residues 165–184 and 366–386 are important for RNA-binding and LLPS (Chong et al., 2018). Aside from the RGG boxes, SERBP1 does not contain any other identifiable RNA-binding motif such as an RNA-recognition motif (RRM), zinc-finger, or K-homology (KH) domain (Maris et al., 2005; Valverde et al., 2008; Cook et al., 2015). We used the PSIPRED (Buchan and Jones, 2019) and DISOPRED3 (Ward et al., 2004) webservers to predict regions of secondary structure and intrinsic disorder respectively in SERBP1 (Figures 1B,C). The results of the PSIPRED algorithm, shown as a cartoon representation (Figure 1B), predicts two regions of secondary structure roughly spanning residues 5–80 and 270–360. These results align well with the DISOPRED3 algorithm predictions which indicate SERBP1 is predominantly disordered, except for the first ∼40 N-terminal residues and residues 285–300 (Figure 1C). Several other segments of SERBP1 approach the cutoff threshold for order (e.g., 230–240, 320–330, 350–365, 395–408) and align well with the PSIPRED secondary structure predictions indicating the potential for transiently-formed structure.

SERBP1 is highly conserved among vertebrates, thus to gain insight into the functional relevance of the different domains of SERBP1, we aligned human SERBP1 to several homologous proteins from invertebrates (Figure 1D). The alignments are shown with gaps, and dashed boxes denote regions with conserved (red) or semi-conserved (blue) sequence homology. Full alignments are presented in Supplementary Figure S1. Notably, the highly homologous regions align well with the predicted secondary structure (Figure 1B) and ordered regions (Figure 1C), in particular residues 285–300, which are predicted to be helical, are highly conserved (region “III” in Figures 1C,D), indicating a potentially crucial role for SERBP1 function. Moreover, it appears that although the two RGG boxes show high conservation among vertebrates, these regions are highly heterogeneous among non-vertebrates (Figure 1D; Supplementary Figure S1). To expand on the role of the RGG boxes for SERBP1 function, we designed two truncated constructs: 149–400 or 189–400 which contain either both (149–400) or only the C-terminal (189–400) RGG box(es) (Figure 1A). In both constructs, we also removed eight C-terminal residues (401–408) that are hydrophobic and contributed to instability and were consistently degraded from SERBP1. To confirm that the truncated proteins faithfully reproduce the structural features of the full-length protein, ¹H,¹⁵N-HSQC spectra were recorded. Overlays of the spectra from full-length SERBP1 with spectra from each of the truncation proteins are almost identical indicating that the truncations did not lead to major structural changes (Supplementary Figure S2). The N-terminal region, that was predicted as structured, was not detected as such in the full-length spectrum, and thus we decided to focus our study on the truncated proteins.

We previously reported the backbone resonance assignments for SERBP1 189–400 (BMRB accession number 50953) and here we report the backbone resonance assignments for SERBP1 149–400 (Supplementary Figure S3). Over 77% of the backbone resonances from the 252 residue SERBP1 149–400 were assigned, excluding the 11 proline residues. Of the 56 residues not assigned, 32 are arginines or glycines that belong to the two RG/RGG repeats and thus, because of the inherent sequence degeneracy in these regions, could not be unambiguously assigned. The remining 24 residues were either ambiguous or were severely overlapped in the spectrum. Overall, assignments of SERBP1 149–400 Cα, Cβ, and C′ chemical shifts were 81, 84.6, and 77.8% complete respectively. Analysis of the Cα, Cβ, C’, H_N, N, and Hα chemical shifts using the Secondary Structure Propensity (SSP) algorithm (Marsh et al., 2006) indicate that the majority of SERBP1 189–400 is disordered, except for residues 289–299, which have significant α-helical character (Figure 2A). A similar analysis for the 149–400 construct revealed α-helical propensity for the same residues (Figure 2B), indicating that the first RGG domain does not influence the formation or stability of the helix. Similar results were obtained with the δ2D (Camilloni et al., 2012) and TALOS-N (Shen and Bax, 2013) algorithms which indicate substantial (>80 %) α-helical propensity for residues 289–299 in both the SERBP1 149–400 and 189–400 constructs (Supplementary Figure S4). Circular dichroic (CD) spectra of SERBP1 149–400 and 189–400 recorded to measure thermal denaturation have a similar appearance and contain features indicative of IDPs such as the strong negative transition at 200 nm (Figure 2C). For both constructs, a negative transition at approximately 222 nm becomes more negative as the temperature is increased from 20 to 80°C. The temperature gradient also reveals an isodichroic point at 212 nm indicative of a two-state transition that could translate to exchange between partially ordered and completely disordered conformations (Greenfield, 2006). Deconvolution of these CD spectra to estimate helical content is complicated by the inherent contributions from polyproline II conformations that contribute to the observed transitions at 200 and 212 nm (Kjaergaard and Poulsen, 2011). Taken together these data are consistent with the chemical shift analysis (Figures 2A,B), secondary structure, and disorder predictions Figures 1B,C and indicate that SERBP1 is primarily disordered with some α-helical character.

To further define the structural properties of SERBP1, we investigated the fast timescale dynamics of the truncation mutants by measuring the ¹⁵N R ₁ and R ₂ relaxation rates, as well as the ¹H-¹⁵N heteronuclear NOE (Figure 3; Supplementary Figure S5). For the 189–400 construct, the average R ₁ and R ₂ values are 1.5 s⁻¹ and 9.6 s⁻¹, respectively, except for residues 289–299 in the region identified as helical by SSP, which have average R ₁ and R ₂ of 1.2 s⁻¹ and 22.5 s⁻¹ respectively. We observed similar average R ₁ and R ₂ (1.5 s⁻¹ and 9.5 s⁻¹ respectively) along with variations in the same region (289–299) for SERBP1 149–400 (Supplementary Figure S5). Average heteronuclear NOE values for both constructs were 0.4 with the residues in the 289–299 stretch displaying values approaching 0.75, indicating the motions of these amino acids are more restricted than the rest of the chain. These data reveal that the two truncated proteins retain virtually identical dynamics and contain a stable α-helix comprising residues 289–299, the stability of which seems to be independent of the RGG boxes.

Size exclusion chromatography (SEC), dynamic light scattering (DLS) and analytical ultracentrifugation (AUC) were employed to assess the oligomeric state of SERBP1 and to determine if the stable α-helix acts as a dimerization interface, as has previously been described for TAR DNA-binding protein (TDP-43) (Conicella et al., 2016). At physiological conditions, SERBP1 189–400 elutes at 14.7 ml (Figure 4A), which, according to column calibration (data not shown) corresponds to a molecular weight of 51.6 kDa, suggesting a possible dimerization of SERBP1 (the molecular weight of monomers is 23.7 kDa) if SERBP1 were a globular, folded protein. However, due to their extended conformations, IDPs are expected to elute earlier in SEC corresponding to larger apparent molecular weights (Uversky, 2012). The SEC experiment was repeated in the presence of 2 M of GdnHCl, resulting in an elution volume of 13.3 ml, corresponding to a molecular weight of 95.9 kDa (Figure 4A). Similar behavior was observed for SERBP1 149–400 which displayed elution volumes of 14.8 and 12.8 ml in absence and in presence of 2 M GdnHCl, respectively (Figure 4B). If SERBP1 formed oligomers in the absence of GdnHCl, the elution volume should increase upon the addition GdnHCl, contrary to the observed decrease. These results suggest that SERBP1 becomes more extended, occupying a much larger steric volume in 2 M GdnHCl than in physiological conditions.

To further assess the oligomeric state of SERBP1, DLS was used to measure the apparent hydrodynamic radius (R _h) at physiological conditions and in the presence of 2 M GdnHCl (Figure 4C). A mean R _h estimation of 3.9 and 4.4 nm under physiological and denaturing conditions, respectively, was obtained for SERBP1 189–400. Notably, the polydispersity in the presence of GdnHCl was ∼30%, higher than the ∼13% that was observed under non-denaturing conditions. The small relative difference in R _h in the presence and absence of GdnHCl indicates that SERBP1 is a monomer at physiological conditions, consistent with the SEC results, adopting a conformation that is more compact than a fully disordered chain. The theoretical R _h value, calculated by the method proposed by Marsh and Forman-Kay (Marsh and Forman-Kay, 2010) was 3.8 nm, in very good agreement with the experimentally measured value of 3.9 nm, further evidence that SERBP1 is monomeric at these conditions (Figure 4C). Finally, AUC experiments were conducted on SERBP1 189–400 in absence and presence of 2 M GdnHCl (Figure 4D). The fitted sedimentation coefficient was lower (1.3 vs. 1.9) and the frictional ratio was higher (2.2 vs. 1.8, not shown) in presence of GdnHCl, indicating SERBP1 189–400 is more compact under physiological conditions than a fully extended polypeptide chain, and that it becomes less compact in denaturing conditions. Similar results were obtained for SERBP1 149–400 with a lower sedimentation coefficient (1.5 vs. 2.2) and a higher frictional ratio (2.1 vs. 1.7, not shown) in the presence vs. absence of 2 M GdnHCl respectively (Supplementary Figure S6). These observations are consistent with the average R ₂ rates recorded for both constructs, (∼9.5 s⁻¹) which are faster than expected for proteins that would preferentially exist in extended conformations (Figure 3B; Supplementary Figure S5B). Together, these data are highly consistent and reveal that SERBP1 is monomeric under the experimental conditions used here, adopting conformations that are more compact than a fully extended polymer.

SERBP1 is thought to be an important regulator of many different mRNAs although the structural details of RNA recognition remain unclear. Previously, the RNAcompete assay defined 5′-GCGCGGG-3′ as the SERBP1 consensus binding motif and subsequently it was shown that an RNA oligonucleotide containing this sequence binds full-length SERBP1 with sub-micromolar affinity (Kosti et al., 2020). Here, heteronuclear NMR was used to study the interaction between SERBP1 189–400 and the 7-mer RNA oligonucleotide. A series of ¹H,¹⁵N heteronuclear single quantum correlation (HSQC) spectra of SERBP1 189–400 were acquired in the presence of increasing concentrations of the 7-mer RNA (Figure 5A). Overall, the spectra remain similar in response to increasing concentrations of the 7-mer indicating the RNA ligand does not induce folding of SERBP1, a phenomenon that has been observed for other IDPs (Sugase et al., 2007; Tompa and Fuxreiter, 2008; Shammas et al., 2016; Bonetti et al., 2018; Robustelli et al., 2020). However, some peaks display strong chemical shift perturbations (CSP), such as A395, S394, and V398, indicating these residues interact strongly with the RNA (Figure 5A, insets). Additionally, glycine peaks (e.g., G363, G366) belonging to the RGG box broaden significantly as the ligand concentration increases, likely due to exchange between the free and the bound forms at an intermediate rate on the NMR time scale. These observations lead to the proposal that electrostatic interactions occur between the positively charged arginine residues of the RGG box and the negatively charged phosphates of the RNA backbone. This encounter complex could then be stabilized by interactions with residues N383-V398, especially with T388 and S394 since serine and threonine are excellent hydrogen bond donors. Plotting the maximal CSP (Δδ_max) against the protein sequence reveals that the RNA interacts with residues spanning from the second RGG box to the C-terminus of SERBP1, with some Δδ_max higher than 0.1 ppm (e.g., N383, T388, S394) (Figure 5B). If an initial SERBP1:RNA encounter complex forms through charge-charge interactions, the complementary RNA sequence (5′-AUAUAAA-3′) may induce similar broadening in the RGG-specific glycine residues. No specific CSPs were observed in the C-terminal region of the SERBP1 189–400 (Figure 5C), and broadening of resonances belonging to the RGG box was not detected (Supplementary Figure S7). Some CSPs higher than 0.05 ppm are randomly dispersed along the chain, but these are more likely indicative of non-specific protein-RNA interactions. Taken together, these data reveal that further experimentation is required to fully elucidate the nature of the SERBP1 RNA recognition and complex formation.

The relative peak intensities of SERBP1 residues before and after titration were plotted as a function of the amino acid sequence to gain insight into the global effect of the RNA binding on the protein. (Figures 5D,E). Uniform broadening of the peaks along the entire protein sequence was observed upon the addition of stoichiometric quantities of the 7-mer RNA, with the exception of residues 250–285, that precede the α-helix. Notably, these residues retain approximately equal intensities in the absence or presence of the RNA (compare Figures 5D,E), suggesting they remain unaffected by interactions with RNA. This region has lower than average heteronuclear NOE (∼0.25) and R ₂ rates (∼7 s⁻¹) indicating that these residues are more dynamic compared to the rest of the protein and remain mobile in the presence of RNA (Figures 3B,C). Finally, no CSPs or peak broadening beyond the uniform broadening described above were observed for residues 289–299 that comprise the α-helix indicating that this region is not involved in recognition or binding of the RNA 7-mer.

Intrinsically disordered RBPs are overrepresented in the group of proteins that are known to participate in biomolecular condensates and undergo LLPS (Burke et al., 2015; Elbaum-Garfinkle et al., 2015; Boeynaems et al., 2017). The phase separation properties of the SERBP1 149–400 and 189–400 constructs were assayed using droplet formation assays and fluorescence recovery after photobleaching (FRAP). At neutral pH, SERBP1 149–400 readily phase separates at a concentration of 10 µM in absence of NaCl. The addition of NaCl abrogates SERBP1 phase separation with noticeably smaller and fewer droplets forming in the presence of 50 mM NaCl and complete dissipation at 150 mM NaCl (Supplementary Figure S8A). Conversely, SERBP1 189–400 does not phase separate at all under the same physiochemical conditions (Supplementary Figure S8B). Increasing NaCl concentrations (50–1000 mM) had no effect on the LLPS characteristics of SERBP1 189–400. Since the main difference between the two constructs is the N-terminal RGG box, it is reasonable to conclude that the RGG boxes promote phase separation of SERBP1, consistent with what has been described for other RGG-containing RBPs (Chong et al., 2018).

Given the observed effects of NaCl on phase separation of SERBP1, the effect of RNA was also tested. In this assay, 150 mM NaCl, 13 % glycerol, and 13% PEG3000 were included in the droplet formation buffer with 12 µM SERBP1 149–400 or SERBP1 189–400 and increasing concentrations of torula yeast RNA (Figures 6A,B). Contrary to the effect of NaCl, RNA promoted phase separation of SERBP1 189–400 at concentrations as low as 0.05 mg/ ml RNA (Figure 6B). Under the three RNA concentrations tested (0.05, 0.2, and 0.4 mg/ ml), the observed average droplet size for SERBP1 189–400 remained consistent at ∼12 μm² (Figure 6D). At the same protein concentration, phase separation of SERBP1 149–400 was promoted by the presence of glycerol and PEG3000 (compare the 0 mg/ ml panels in Figures 6 A,B) while droplet size strongly correlated to the RNA concentration (Figure 6A). At no added RNA or 0.05 mg/ ml RNA, the average observed droplet size for SERBP1 149–400 was ∼50 μm². The average droplet size significantly decreased to ∼20 μm² as the RNA concentration increased to 0.2 and 0.4 mg/ ml (Figure 6C). This trend held when the experiment was repeated with 6 µM SERBP1 149–400 albeit the average observed droplet size was lower, ranging from 10 μm² without RNA to 2 μm² at an RNA concentration of 0.4 mg/ ml (Supplementary Figure S9). RNA modulates SERBP1 phase separation, promoting phase separation for SERBP1 189–400 under non-permissive conditions, as well as altering the droplet characteristics formed from SERBP1 149–400. While the influence of crowding agents cannot be separated from the contribution of the RGG boxes to phase separation in these experiments, taken together these results show that the RGG boxes are important for SERBP1 LLPS, which likely proceeds through a charge-mediated mechanism.

The liquid -like nature of the condensates formed by SERBP1 149–400 in the presence and absence of RNA were probed using FRAP experiments (Figure 6D). In the absence of RNA, the recovery of fluorescence plateaus at ∼70 s, while in the presence of 0.05 mg/ ml of RNA, the fluorescence intensity recovers faster, reaching a plateau in ∼40 s. The faster recovery of fluorescence in the presence of RNA is indicative of faster diffusion and hence more dynamic droplets. These results support the droplet formation assays suggesting that the two RGG boxes might interact synergistically with the RNA to modulate the condensate behavior of SERBP1. Further investigation will be necessary to decipher the molecular mechanisms of SERBP1 LLPS.