The homologs of human DRG1 and DRG2 were identified in a variety of organisms at the National Center for Biotechnology Information database (NCBI) using the blastp algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Genomes were additionally searched in the Ensembl database (https://metazoa.ensembl.org/index.html) and the TAIR database (https://www.arabidopsis.org/index.jsp) for Mnemiopsis leidyi and Arabidopsis thaliana drg genes, respectively. Protein sequences from selected organisms (listed in Supplementary Table S2) were subjected to a multiple sequence alignment analysis conducted by the MUSCLE algorithm58. To resolve the phylogenetic relationships of the DRG subfamily of proteins among archaea and eukaryotes, a maximum-likelihood tree was estimated in the MEGA7 software59. Unlike the eukaryotes, archaea contain only one DRG that shows a similar identity with both human DRG proteins, hence it was used as an outgroup to root a tree. Maximum-likelihood tree was based on LG + G + I evolutionary model60, according to the results obtained by ProtTest61. To evaluate the robustness of the phylogenetic tree, a bootstrap analysis from 1000 replications was performed. Obtained multiple sequence alignment was used to generate an amino acid identity and similarity matrices via the Matrix Global Alignment Tool (MatGAT2.01 with BLOSUM62 scores62), presented in Supplementary Table S3. The summarized identity/similarity datasets of DRG1 proteins were visualized using a heat map conducted by Morpheus (https://software.broadinstitute.org/morpheus/). For the intron-mapping of drg1 genes from selected metazoan species and choanoflagellate, nucleotide sequences (with indicated intron positions) were taken from the NCBI’s genomic database (https://www.ncbi.nlm.nih.gov/genome/). The exact position and the phase of each intron was verified by manually.
The AutoDock Vina software was utilized in the docking calculation, and as a docking target we used the structure of EsuDRG1 predicted with the Alpha fold38. The docking target was previously protonated using the MolProbity63. The GTP structure was downloaded from the ZINC database64 and prepared using Ligprep from the Schrödinger program package (Schrödinger Release 2021-4: LigPrep, Schrödinger, LLC, New York, NY, 2021). The active site of the target DRG1 protein was treated as rigid except residues: S72, S76, K75, T77, N246, K247, D249, H271, Q250, which presumably interact with the GTP molecule. The grid size was set at 30 × 30 × 30 XYZ points centered at (-13.877, 16.691, -9.281) Å and the grid spacing was set to 1 Å. The docking was carried out with Autodock Vina39. The target and ligand final preparation, visualization of the grid box, and the docking results were done by utilizing the AutoDock tools65. The generated protein:ligand complex was geometrically optimised in Maestro from Schrödinger program package (Schrödinger Release 2021-4: Maestro, Schrödinger, LLC, New York, NY, 2021). Structure visualization and analysis were performed using UCSF Chimera66.
Sponge sampling was performed with the permit from the Ministry of Economy and Sustainable Development, Croatia.
The tissue from the sponge Eunapius subterraneus (Tounjčica cave, Croatia) was homogenized and the cells were isolated using Falcon 200, 100 and 40 µm Cell Strainers. Total RNA was subsequently isolated using the RNeasy Kit (QIAGEN) according to the manufacturer’s instructions. The cDNA library was prepared from total RNA isolated from E. subterraneus using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems™) according to the manufacturer’s protocols.
Unpublished transcriptome of E. subterraneus for the homologs of human DRG1, DRG2 and DFRP1 was searched. Based on the identified sponge sequence, primers for the sponge DRG1 (EsuDRG1, OL692370) and DFRP1 (EsuDFRP1, OL692371) from the cDNA library (Supplementary Table S1) were designed. EsuDRG1 and EsuDFRP1 were amplified, sequenced and cloned into pET28b, pEGFP-N1, pmCherry-C1, and pcDNA3.1 vectors. The cDNA sequences of human DRG1 (HsaDRG1) and DFRP1 (HsaDFRP1) from commercially available products were cloned into the same vectors. The primers and restriction enzymes used for cloning of EsuDRG1, HsaDRG1, EsuDFRP1 and HsaDFRP1 are listed in Supplementary Table S1. The resulting constructs are His-, GFP-, CHERRY-, FLAG-, MYC-tagged depending on the experiment.
Recombinant sponge and human proteins were produced in the E.coli strain BL21 CodonPlus (DE3). Cells transformed with sponge or human pET28b-DRG1-His and pET28b-DFRP1-His were grown at 37 °C in TB/Kan medium to OD600 of 0.6, induced with 0.1 mM IPTG, and grown at 16 °C for 18 h. Afterwards, the cells were washed and incubated on ice for 30 min in lysis buffer (50 mM HEPES, pH 7.4, 400 mM NaCl, 5 mM MgCl2, 10% glycerol (v/v), 10 mM imidazole, 1 mg/mL lysozyme (Sigma-Aldrich), protease inhibitor cocktail (Roche Applied Science) and 25000 units/mL Benzonase® Nuclease (Sigma-Aldrich)) and sonicated for 8 × 30 s at 4 °C. The lysate was purified by centrifugation for 40 min at 13280×g and 4 °C and filtered through a 0.22 μm sterile membrane filter. The filtered solution was loaded onto a cobalt affinity resin column (Takara). His tagged proteins were eluted with 150 mM imidazole and concentrated in storage buffer (25 mM HEPES pH 7.4, 300 mM NaCl, 5 mM DTT, and 10% glycerol) using Amicon 10 kDa cutoff filters (Merck). Produced proteins were analysed by SDS–polyacrylamide gel electrophoresis.
Size-exclusion chromatography was performed at Biocentar d.o.o., Zagreb, Croatia. Recombinant proteins (EsuDRG1-His, EsuDFRP1-His and HsaDRG1-His) were loaded onto size-exclusion Superdex 200 Increase 10/300 GL column (GE Healthcare), pre-equilibrated with 10 mM phosphate buffer, 140 mM NaCl, pH 7.4. Proteins were eluted at 0.5 mL/2 min or 0.5 mL/1 min using an Äkta avant 25 system (GE Healthcare) at 4 °C. Injection volume was 500 µL. The column was calibrated with Bio-Rad Gel filtration standards: Thyroglobulin (670 kDa), γ-globulin (158 kDa), Ovalbumin (44 kDa), Myoglobin (17 kDa), and Vitamin B21 (1.35 kDa).
The intrinsic GTPase activity of EsuDRG1 and HsaDRG1 recombinant proteins was measured using the GTPase-Glo Assay (Promega) according to the manufacturer’s guidelines. For optimization, the purified proteins were serially diluted in a GTPase/GAP buffer containing 2 µM GTP, and the assay was carried out for 120 min at 37 °C. Luminescence was measured using white flat bottom 384-well microplates (Greiner) on the Infinite M200 plate reader (Tecan). To investigate the effect of DFRP1 on DRG1 GTPase activity, an equimolar mixture of 1.2 μM EsuDRG1 and EsuDFRP1 was assayed as described and relative luminescence was measured.
The DNA binding ability of EsuDRG1 and HsaDRG1 was assayed in vitro as described30,67,68. Briefly, the reactions contained 200 ng of single-stranded circular DNA of bacteriophage ΦX174 (NEB, #N3023S) or double-stranded covalently closed circular form of ΦX174 (NEB, # N3021S). The amount of purified proteins in the reaction ranged from 100 to 1600 ng. The reactions were performed in 20 μL, containing 40 mM Tris–acetate (pH 7.5) and 1 mM EDTA incubated for 30 min at 37 °C. The products were analysed by gel electrophoresis at 3 V/cm in 0.5% agarose for 3 h in 40 mM Tris–acetate (pH 7.5), 1 mM EDTA running buffer.
The nonspecific RNA binding ability of DRG1 proteins was assayed in vitro as described7,69. Briefly, 0.5 mg of proteins were incubated in 100 µL of cold reaction buffer (10 mM HEPES–NaOH (pH 7.4), 100 mM NaCl, 2 mM MgCl2, 0.1% Triton X-100, 3 mM DTT) with a free poly(U) (Sigma-Aldrich) at concentrations of 0, 0.1 and 1 mg/mL. The reactions were incubated for 30 min at 4 °C in the rotator. Subsequently, 10 µL of 50% poly(U)-agarose (Sigma Aldrich) in binding buffer was added to each reaction mixture and incubated for 30 min at 4 °C. The beads were washed six times in binding buffer following centrifugation for 1 min at 4 °C. Finally, proteins bound to the poly(U)-agarose were eluted by adding 10 µL of 4 × SDS sample buffer and boiled at 95 °C for 6 min. Samples were loaded onto a 12% SDS-PAGE gel and visualized by Coomassie brilliant blue.
Protein cross-linking with glutaraldehyde was performed as described30,69 with the following modifications. The reactions containing 1 μg of DRG1 proteins alone or a 2 μg mixture of each of the EsuDRG1 and EsuDFRP1 protein were preincubated in PBS for 15 min. DRG1 proteins were crosslinked with 0.063%, 0.0125% and 0.025% glutaraldehyde for 5 min, and DRG1 and DFRP1 mixture was incubated with 0.125%, 0.25% and 0.5% glutaraldehyde for 30 min. The reactions were quenched with 0.2 M Tris–HCl, pH 7.5 for 15 min. All steps were performed at room temperature. The reaction products and untreated proteins (control) were boiled at 95 °C for 5 min, loaded onto 12% SDS-PAGE gel and visualized by staining with Coomassie brilliant blue. The same was repeated with human homologs.
Human breast/mammary cancer cells MCF-7 (ECACC cat. no. 86012803) and MDA-MB-231 (ATCC cat. no. HTB-26) and cervical cancer cells HeLa (ATCC cat. no. CCL-2) were maintained in the Dulbecco’s Modified Eagle Medium with high glucose (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, Capricorn Scientific), 1% nonessential amino acids (Sigma-Aldrich) and 1% antibiotic/antimycotic solution (Capricorn Scientific) in the humidified chamber at 37 °C with 5% CO2. Twenty-four hours after seeding, cells were transfected using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s protocol and incubated for additional 24 h.
For protein level analysis, 5 × 105 of MCF-7 cells were seeded in a six-well plate, transfected with expression vector pcDNA3 for FLAG-tagged sponge (EsuDRG1-FLAG) or human DRG1 (HsaDRG1-FLAG) and/or MYC-tagged sponge (EsuDFRP1-MYC) or human DFRP1 (HsaDFRP1-MYC), respectively. For Western blot analysis, cell lysates were prepared as follows: the cells were washed three times in PBS (pH 7.5) and homogenized in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP40, 0.1% SDS, 0.5% sodium deoxycholate) containing a protein inhibitor cocktail (Roche Applied Science) and centrifuged at 16 000 × g for 10 min at 4 °C. The total protein concentration was measured using the commercially available Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Cell lysates were mixed with 6 × sample buffer (60% glycerol, 12% SDS, 3% DTT, 1/8 v/v 0.5 M Tris pH 6.8, bromophenol blue) and heated at 70 °C for 10 min. Equivalent amounts of protein were loaded onto Tris–Glycine gels. After SDS-PAGE, proteins were electrotransferred onto a PVDF membrane (Roche Applied Science) and incubated in primary antibody solution. Exogenous proteins were detected with anti-FLAG (clone M2, Sigma-Aldrich) and anti-MYC (clone 9E10, Sigma-Aldrich) antibodies, and endogenous proteins were detected with polyclonal antibodies against DFRP1 (Thermo Fisher Scientific) and DRG1 (Abcam) and monoclonal α-tubulin antibody (clone DM1A, Sigma-Aldrich). After incubation with primary antibodies, the membranes were washed and incubated in the HRP-conjugated secondary antibody (Bio-Rad) solution. Proteins were visualized by chemiluminescence using ECL blotting substrate (GE Healthcare) on a documentation system from UVItec Cambridge. AmidoBlack (Sigma-Aldrich) staining of membranes was used as a loading control, according to the manufacturer’s protocol. Protein signals were quantified using the ImageJ software (National Institutes of Health).
For immunocytochemistry, MCF-7 (5 × 104 cells/well) and HeLa cells (2 × 104 cells/well) were seeded on sterile glass coverslips in a 24-well plate to achieve 70% confluence. After 24 h, cells were co-transfected with the fluorescently labelled sponge (EsuDRG1-GFP) or human DRG1 (HsaDRG1-GFP) together with sponge (EsuDFRP1-CHERRY) or human DFRP1 (HsaDFRP1–CHERRY). Control cells were co-transfected with empty-GFP (pEGFP-N1) and empty-CHERRY (pmCherry–C1) vector, respectively. Immunocytochemistry on MCF-7 cells was performed as previously described70. In short, the cells grown on coverslips were washed three times in PBS, fixed with 4% sucrose/paraformaldehyde for 15 min and permeabilized with 0.2% saponin in PBS. The cells were blocked in 4% donkey serum (Sigma-Aldrich) for 1 h at room temperature and subsequently incubated overnight with the primary antibody against α-tubulin (clone DM1A, Sigma-Aldrich) diluted in a blocking solution, followed by incubation in fluorescently labelled secondary antibody (Thermo Fisher Scientific) for 1 h. Hoechst (Sigma-Aldrich) was used to counterstain nuclei. Confocal images were acquired using the laser scanning confocal microscope Leica TCS SP8 (Leica Microsystems, Wetzlar, Germany). Additional image processing was performed by the ImageJ software (National Institutes of Health).
For detection of DRG1 and DFRP1 complexes, 5 × 105 of MCF-7 cells were seeded in a six-well plate, co-transfected with EsuDRG1-FLAG or HsaDRG1-FLAG and EsuDFRP1-MYC or HsaDFRP1-MYC. Briefly, cells were collected 24 h after transfection, lysed in coIP buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP40, 0.5% Triton X-100), supplemented with protease inhibitor cocktail (Roche Applied Science) and centrifuged at 16 000 × g for 10 min at 4 °C. Total protein concentration was measured using the commercially available Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Immunoprecipitation of FLAG-tagged EsuDRG1 or HsaDRG1 was performed using the anti-FLAG M2 affinity gel (Sigma-Aldrich) according to the manufacturer’s protocol. Briefly, 40 µL of gel was washed and incubated with 200 µg of proteins on a rotator at 4 °C overnight. The next day, the samples were centrifuged at 800 × g for 15 min at 4 °C, supernatant was saved, and the complexes were washed two times in a buffer containing 50 mM Tris pH 7.6, 500 mM NaCl, 2 mM EDTA, and two times in buffer with 50 mM Tris pH 7.6, 150 mM NaCl, 2 mM EDTA. Between each washing step, samples were centrifuged at 800 × g for 1 min at 4 °C. Complexes were resuspended in a sample buffer (20% glycerol, 4% SDS, 1% DTT, 1/24 v/v 0.5 M Tris pH 6.8, bromophenol blue), separated by SDS-PAGE and electrotransferred onto PVDF membrane (Roche Applied Science). DRG1 and DFRP1 complexes were detected using the primary antibodies against FLAG (clone M2, Sigma-Aldrich) and MYC (clone 9E10, Sigma-Aldrich), and visualized by chemiluminescence using ECL blotting substrate (GE Healthcare) on a documentation system from UVItec Cambridge.
Cell proliferation was monitored using the MTT assay. Briefly, 7 × 103 of MCF-7 cells and 4 × 103 of MDA-MB-231 cells were seeded in a 96-well plate and transfected with EsuDRG1-FLAG and/or HsaDRG1-FLAG and/or EsuDFRP1-MYC or HsaDFRP1-MYC. Forty-eight h after the transfection growth medium was removed, 1 × MTT was added and cells were incubated for 4 h in the growth conditions, followed by an addition of dimethyl sulphoxide and 2 min incubation with gentle mixing. The absorbance was measured at 570 nm on ELISA microplate reader (LabSystem Multiskan MS, Artisan Technology Group).
To test colony formation, MDA-MB-231 cells were co-transfected with EsuDRG1-FLAG or HsaDRG1-FLAG and EsuDFRP1-MYC or HsaDFRP1-MYC. Twenty-four h after the transfection cells were resuspended and seeded in 60 mm dishes at 5 × 104 cells/dish and G418 (Neomycin, Sigma-Aldrich) was added to a final concentration of 500 µg/mL for the selection of resistant colonies. After 10 days, the resistant colonies were fixed with 100% methanol for 10 min, dried, stained with 10% Giemsa (Sigma-Aldrich) for 30 min and counted.
The MDA-MB-231 (5 × 104 cells/well) cells were seeded in a 24-well plate, co-transfected with EsuDRG1-FLAG or HsaDRG1-FLAG and EsuDFRP1-MYC or HsaDFRP1-MYC. Twenty-four h after transfection, a wound by scratching the cell monolayer in a straight line was made with a sterile 100 μL tip. The cells were washed with fresh medium and incubated for 24 h. Cell migration was monitored by measuring the distances between the two margins of the scratch after 24 h in five fields of each chamber using 100× magnification on the microscope (Olympus CKX41, Tokyo, Japan). Distances were quantified by comparing the distances at the time point zero and after 24 h in the same field using the ImageJ software (National Institutes of Health).
For monitoring cell migration MDA-MB-231 cells were co-transfected with EsuDRG1-FLAG or HsaDRG1-FLAG and EsuDFRP1-MYC or HsaDFRP1-MYC. The cells were seeded in a migration Transwell cell culture inserts (pore size 8 mm; Corning) at a density of 2.5 × 104 cells/well, and left to migrate for 24 h towards 10% FBS in DMEM as a chemoattractant. The cells that migrated to the underside of the filter were fixed with 4% PFA and stained with 1% crystal violet solution. Images were acquired at a 200× magnification using the microscope (Olympus BX51, Tokyo, Japan) and quantified by the ImageJ software (National Institutes of Health, USA).
All biological experiments were repeated at least three times in biological duplicates or triplicates. Statistical analysis was performed using the SPSS statistical package for Windows, v17. For the determination of statistically significant differences between the means of three or more groups, one-way ANOVA with appropriate post-hoc analyses was used. To determine if a difference exists between the means of two independent groups, the t-test was performed. The statistical significance of the tests was set at p < 0.05.
All authors read and approved the final manuscript. All authors confirmed that submitted manuscript has neither been published, nor simultaneously submitted elsewhere.
