Posttranscriptional modification of tumor-associated factors plays a pivotal role in breast cancer progression. However, the underlying mechanism remains unknown. M6A modifications in cancer cells are dynamic and reversible and have been found to impact tumor initiation and progression through various mechanisms. In this study, we explored the regulatory mechanism of breast cancer cell proliferation and metabolism through m6A methylation in the Hippo pathway. A combination of MeRIP-seq, RNA-seq and metabolomics-seq was utilized to reveal a map of m6A modifications in breast cancer tissues and cells. We conducted RNA pull-down assays, RIP-qPCR, MeRIP-qPCR, and RNA stability analysis to identify the relationship between m6A proteins and LATS1 in m6A regulation in breast cancer cells. The expression and biological functions of m6A proteins were confirmed in breast cancer cells in vitro and in vivo. Furthermore, we investigated the phosphorylation levels and localization of YAP/TAZ to reveal that the activity of the Hippo pathway was affected by m6A regulation of LATS1 in breast cancer cells. We demonstrated that m6A regulation plays an important role in proliferation and glycolytic metabolism in breast cancer through the Hippo pathway factor, LATS1. METTL3 was identified as the m6A writer, with YTHDF2 as the reader protein of LATS1 mRNA, which plays a positive role in promoting both tumorigenesis and glycolysis in breast cancer. High levels of m6A modification were induced by METTL3 in LATS1 mRNA. YTHDF2 identified m6A sites in LATS1 mRNA and reduced its stability. Knockout of the protein expression of METTL3 or YTHDF2 increased the expression of LATS1 mRNA and suppressed breast cancer tumorigenesis by activating YAP/TAZ in the Hippo pathway.In summary, we discovered that the METTL3-LATS1-YTHDF2 pathway plays an important role in the progression of breast cancer by activating YAP/TAZ in the Hippo pathway.The online version contains supplementary material available at 10.1186/s13046-022-02581-1. The latest global burden of cancer released by the International Agency for Research on Cancer (IARC) in 2020 declared breast cancer to be the world's leading cancer . Owing tNonmutational epigenetic reprogramming is one of the 14 features of cancer listed in the third edition of Hallmarks of Cancer . As the The Hippo pathway plays an important role in organ development and serves as a tumorigenesis suppressor. Following stimulation with extracellular growth inhibition signals, a series of kinase cascades, such as LATS1/LATS2, are activated, resulting in phosphorylation of the effector factor Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Notably, recent findings have implicated YAP and TAZ in the metabolism of cancer cells . The HipWhether m6A modification occurs in the Hippo pathway remains unclear. Recent studies have shown that METTL3 is highly expressed in breast cancer  and can After obtaining adequate informed consent, breast cancer tissue and adjacent normal tissue were obtained from 8 patients who underwent curative resection for breast cancer at Nanfang Hospital of Southern Medical University, between November 2019 and May 2020. All patients satisfied the following inclusion criteria: the surgical margins were confirmed to contain no residual carcinoma tissue; clinicopathological information on age, sex, clinical stage, neoadjuvant therapy, ER, PR, HER2, Ki67, histological subtype and histological grade was available and is listed in Supplementary Table Human breast cancer cell lines  were purchased from American Type Culture Collection . MCF-7 cells were cultured in MEM  supplemented with 10% fetal bovine serum , 1% penicillin‒streptomycin (Gibco), 10 μg/ml insulin  and 1⨯ nonessential amino acids . SK-BR-3, MCF-7, T47D, BT474, MDA-MB-468, and MDA-MB-231 cells were cultured in RPMI 1640  supplemented with 10% fetal bovine serum  and 1% penicillin‒streptomycin (Gibco).For CRISPR‒Cas9 cells, 100 μM sgRNA Top/Bottom Oligo solution was configured with ddH2O, and the sgRNA sequences were as follows: sgMETTL3:5’-ATTCTGTGACTATGGAACCA-3’, sgYTHDF1 5’-AGTTTCAAAGCCGACCTCGT-3’, sgYTHDF2 5’-GTCCATTACTAGTAACATCG-3’, and sgYTHDF3: CAACCGAAACTTAAACCCAA-3’. PCR was used to produce linked products that were transformed into DH5α cells. Lentiviral overexpression plasmids for human METTL3 and YTHDF2 were transfected into cells as previously described . Cells wLysis buffer was used for total protein analysis of extracted cells. Samples were separated on an 8–15% gel by SDS-PAGE. Nitrocellulose membranes were blocked with blocking buffer and incubated with the appropriate primary antibody. The membranes were washed with blocking buffer three times, probed with the appropriate secondary antibody and developed using SuperSignal West Pico or Dura (Thermo Fisher Scientific).Real-time PCR analysis was performed by using the Biosystems Step-One-Plus Real-Time PCR System with FastStart Universal SYBR Green Master Mix (Roche). The primer sets used were as follows.GAPDH-F: GAACGGGAAGCTCACTGG,GAPDH-R: GCCTGCTTCACCACCTTCT,METTL3-F: AGATGGGGTAGAAAGCCTCCT,METTL3-R: TGGTCAGCATAGGTTACAAGAGT,LATS1-F: AATTTGGGACGCATCATAAAGCC,LATS1-R: TCGTCGAGGATCTTGGTAACTC,YTHDF2-F: TAGCCAACTGCGACACATTC, andYTHDF2-R: CACGACCTTGACGTTCCTTT.The PCR conditions were as follows: 10 min at 95 °C and 40 cycles of 15 s at 95 °C and 1 min at 60 °C. The average Ct value for each gene was determined from triplicate reactions and was normalized as previously reported.6 cells per well. After incubation for 24 h at 37 °C, 1 ml medium was added to each well and incubated for 2 h after discarding the old medium. The cells were collected in flow tubes and centrifuged at 1,500 rpm for 5 min. The supernatant was discarded and the cells were resuspended in PBS and centrifuged at 1,500 rpm for 5 min. After discarding the supernatant, the cells were fixed with 4% paraformaldehyde for 15–30 min, neutralized with glycine for 5 min, and resuspended in PBS. The cells were then incubated with 0.5% Triton X-100 penetrant at room temperature for 10 min. One hundred microliters of 1⨯ Apollo staining reaction solution was added to each tube and the cells were resuspended again. After incubation for 10 min at room temperature, the staining reaction solution was discarded by centrifugation at 1,500 rpm for 5 min. Next, 0.5% Triton X-100 penetrant was used to clean the cells 1–3 times at room temperature and the cells were resuspended in PBS. Flow cytometry was performed immediately after staining.For the EdU cell proliferation assay, the cells were trypsinized and seeded onto 6-well plates at a density of 1 × 106) were implanted into the subcutaneous axilla of the forelimb of 3–4-week-old BALB/c nude mice. Seven days after transplantation, the diameter of the tumors was measured, and the tumors were removed after three weeks.All animal experiments were approved by the Institutional Animal Care and Use Committee of Southern Medical University. Breast cancer cells , according to the manufacturer’s protocol. The quantity and integrity of the RNA yield were assessed using a K5500 microspectrophotometer  and the Agilent 2200 TapeStation system , respectively. m6A antibody-immunoprecipitated RNA was quality controlled using the Qubit  and Agilent 2200 TapeStation  systems. Briefly, RNA was fragmented into molecules approximately 200 bp in length. The RNA fragments were then subjected to first-strand and second-strand cDNA synthesis, followed by adaptor ligation and enrichment with a low cycle according to the instructions of the NEBNext® Ultra RNA LibraryPrep Kit for Illumina . The final library product was assessed using the Agilent 2200 TapeStation and Qubit® system , and then sequenced on an Illumina platform  with a paired-end length of 150 bp at RiboBio Co., Ltd. . Adaptor and low-quality bases were trimmed using Trimmomatic tools (version: 0.36), and the clean reads were subjected to rRNA deletion through RNAcentral to obtain effective reads. Genomic alignment was performed using TopHat (version 2.0.13) to obtain uniquely mapped reads. Effective reads from the input sample were used for RNA-seq analysis and the read count value of each transcript was calculated using HTSeq (version 0.6.0). Differentially expressed genes were identified using the DEseq/DESeq2/edgeR/DEGseq R package according to the following criteria: |log2 (fold change)|≥ 1 and SRAMP is a site prediction tool based on a random forest machine-learning framework that can predict m6A sites based on sequence-derived features. The m6A locus was predicted using the target sequence .A high-resolution mass spectrometer  was used to collect positive ion (POS) and negative ion (NEG) data from 18 cell samples for untargeted metabolomics detection using LC‒MS/MS technology to explore the metabolomic composition and biological function of the samples. Compound Discoverer 3.1.0  software was used for data processing, including peak extraction, peak alignment, fill gaps, and compound identification. An in-house metabolome information analysis process was used to carry out metabolite annotation, classification , and enrichment analysis for the identified substances, and to explain the physical and chemical properties and biological functions of the metabolites. The R software package metaX  and StudTotal RNA was extracted using TRIzol reagent  as previously reported . Total RCells in the treatment and control groups were collected after 24 h. RNA was extracted using TRIzol reagent, and mRNA expression was determined by qPCR.2 at 37 °C. The required drugs and Seahorse XF basic culture medium were prepared simultaneously, and the pH of the medium was adjusted to 7.4. The cells were then placed in a 37 °C water bath for 1 h before use. On the second day, the cells were washed twice with Seahorse XF basic culture medium in a water bath and 175 μl Seahorse XF basic culture medium was added to each well. The cells were then cultured in a CO2-free incubator at 37 °C for 1 h. Each drug was diluted to the required concentration, and 25 μl was added to each well of an XF96 extracellular flux assay kit. Thirty minutes later, the lower part of the XF96 Extracellular Flux Assay Kit was removed and replaced with cell plates that had been cultured in a CO2-free incubator at 37 °C for 1 h and assessed with a computer.On the same day that the cells were seeded, 180 μl of hydration solution was added to the lower part of the XF96 Extracellular Flux Assay Kit. Cells were hydrated overnight in an incubator without COEndogenous RNA was retrieved after trapping in the nucleus or cytoplasm with an antibody or epitope marker. The RNA-binding protein was separated from the bound RNA by immunoprecipitation. The cells were crosslinked with 1% formaldehyde and treated with 150 mM NaCl RIPA buffer containing RNase and protease inhibitors. Cells were lysed in 0.5% sodium deoxycholate, 0.1% SDS, 1% NP40, 1 mM EDTA, and 50 mM Tris (pH 8.0) for 30 min and centrifuged for precipitation. The supernatant was incubated four times with METTL3, YTHDF1, or YTHDF2 primary antibody or the corresponding IgG antibody for 4 h, and protein A/G glycosylated beads were added and incubated for 2 h with shaking. After washing three times with RIPA buffer, RNA was extracted after crosslinking. Quantitative RT-PCR was performed to measure RNA.RNA pulldown assays were performed in accordance with the Pierce™ Magnetic RNA‒ Protein Pull-down Kit protocol. RNA was prepared by in vitro transcription and biotin was used. Poly (A) 25 RNA was used as the negative control. Total protein was extracted from each group, and the required protein concentration was greater than 2 mg/ml. RNA pull-down was performed according to a standard protocol, and the expression level was confirmed by western blotting.p < 0.05 was considered significant. Two patient cohorts were compared using a Kaplan–‒Meier survival plot, and log-rank p value were calculated.Data were analyzed using SPSS 20.0. With a two-tailed independent Student’s t-test, www.ncbi.nlm.nih.gov/geo/). By analyzing the sequencing data, we found that large amounts of differentially methylated or modified mRNAs existed in cancer tissue , a database for predicting the target genes of m6A writers, erasers, and readers, was used to search for proteins related to m6A modification of LATS1 mRNA. As shown in Fig. To investigate m6A modification levels in breast cancer tissue samples, MeRIP-seq was conducted for paired breast ductal cancer tissues and adjacent normal breast tissue samples. The corresponding sequencing data have been uploaded to the GEO database  Venn diagram (left panel) of common (the intersection area) and unique peaks (non-intersection region) of another pair of breast cancer tissue compared with adjacent normal tissue, |log2(fold change)|>1 and q value<0.05. The gene region (right panel) annotated by difference Peaks of breast cancer tissue compared with adjacent normal tissue, |log2(fold change)|>1 and q value<0.05. (b) Differential m6A and differential expression combined analysis diagram of another pair of breast cancer tissue compared with adjacent normal tissue. (c) GO analysis results of differential methylated genes. Differential methylated genes were obtained by TCGA clinical data of breast cancer and compared with MeRIP- seq data. (d) Enrichment analysis result of biological progress (BP) of differential methylated genes. (e) Location map of LATS1 in differential methylated KEGG pathway of Hippo. (f) Volcano map of differentially expressed genes based on transcriptome sequencing of two pairs of breast cancer tissue compared with adjacent normal tissue. (g) Differentially expressed genes of Hippo pathway in transcriptome sequencing of breast cancer tissues compared with adjacent normal tissues, n=2. (h) M6A peaks on LATS1 mRNA originated from MeRIP-seq. M6A peaks were visualized by IGV. The red peaks show the results of MeRIP and the blue peaks represent the input. Supplementary Figure 2. Expression of m6A proteins in breast cancer tissue and cells. (a) The correlation between METTL3 and the expression level of ER/ PR/ HER2 in breast adenocarcinomas . (b) The correlation between METTL3 protein expression and the prognosis of Luminal A (upper panel) and Luminal B (lower panel) breast adenocarcinomas. (c) Alter the mRNA expression level of METTL3 in MCF-7 and T47D cells, ** p<0.01. (d) Alter the protein level of METTL3 in MCF-7 and T47D cells. Supplementary Figure 3. METTL3 promotes tumorigenesis of T47D cells. (a) The proliferation of T47D was determined with EdU cellular proliferation assay with flow detection after altering the expression of METTL3, n=3, * p <0.05, ** p <0.01. (b) The apoptotic cell number was counted with fluorescein assay when knocking out or overexpressing METTL3 in T47D, n=3, *** p <0.001. (c) Migration ability of T47D was suppressed when knocking out the expression of METTL3 detected at 0h, 12h, 24h and 36h. (d) Cell invasion ability was inhibited after blocking the expression of METTL3 in cells detected by trans-well assay, n=3, ** p <0.01. (e) The tumorigenesis of T47D in nude mice was significantly suppressed when knocking out the expression of METTL3, n=7, * p <0.05, *** p <0.001. Supplementary Figure 4. Large amounts of differently expressed metabolites were involved in the deletion of METTL3 in MCF-7. (a) Heat map of differentially expressed metabolites when knocking out the expression of METTL3 in MCF-7, n=6. (b) KEGG enrichment results of differential metabolites affected by METTL3 deletion in MCF-7. Supplementary Figure 5. METTL3 promotes glycolysis and proliferation of T47D cells. (a) The ECAR change between METTL3 KO group and control group of T47D cells in response to glucose, oligomycin and 2-DG, n=4, ** p <0.01. (b) The level of glycolysis, glycolysis reserve and glycolysis capacity in T47D cells with METTL3 knock-out and control group, n=4, * p <0.05, *** p < 0.001. (c) The protein level (left panel) and mRNA level (right panel) of LATS1 after knocking out and overexpressing METTL3 in MCF-7 and T47D cells, n=3, * p <0.05, ** p < 0.01. (d) MeRIP-qPCR shown the m6A level of LATS1 mRNA after altered the expression of METTL3 in T47D, n=3, ** p <0.01. (e) MeRIP-qPCR revealed the m6A level and METTL3 binding level of LATS1 mRNA when muted the m6A methylation sites on LATS1 mRNA in T47D, n=3, ** p <0.01, *** p < 0.001. (f) Rescued the expression of LATS1 with knocking out the expression of METTL3 and siRNA of LATS1 remarkably affected the proliferation of T47D cells. (g) The glycolysis was regained by rescuing the expression of LATS1 by METTL3 in T47D cells, n=4, *** p < 0.001. Supplementary Figure 6. YTHDF2 helped promote the tumorigenesis of T47D breast cancer cells. (a) Knocking out YTHDF2 remarkably suppress the glycolysis progress in T47D cells, n=4, *** p <0.001. (b) Deleting the expression of YTHDF2 significantly inhibited the tumor growth in vivo, n=7, ** p <0.01, *** p <0.001. (c)The colony formation ability of T47D cells was suppressed by the deletion of YTHDF2 and reversed by downregulated the expression of LATS1 at the same time. (d) YTHDF2 knock out led to the slower proliferation of T47D cells and such inhibition could be rescued by LATS1 knockdown, n=3, ** p <0.01. (e) YTHDF2 deletion increased the apoptosis rate of T47D cells, n=3, ** p <0.01, *** p <0.001. (f) Trans-well assay revealed the invasion ability of T47D could be controlled by missing expression of YTHDF2, but also could be rescued by LATS1 siRNA, n=3, * p <0.05, ** p <0.01.Additional file 2:Supplementary Table 1. Data of the included patients. Clinicopathological information on age, sex, clinical stage, neoadjuvant therapy, adjuvant therapy, ER, PR, HER2, Ki67, histological subtype and histological grade of each included patient. Supplementary Table 2. Differential m6A sites in two groups. Specific sites of 1850 up regulated and 1807 down regulated genes of breast cancer cells, p-values <0.05 were indicated. Supplementary Table 3. The relationship between METTL3 and patient clinicopathological features. The age of patients was significantly related to the expression of METTL3, p-values <0.05 were indicated. Supplementary Table 4. KEGG analysis of MCF-7 between METTL3 KO group and control group. KEGG pathways of differentially expressed genes that were altered significantly after METTL3 knockout, p-values <0.05 were indicated. Supplementary Table 5. Metabolites involved in METTL3 knockout in MCF-7. KEGG pathways of different metabolites that were altered significantly after knocking out METTL3 expression, p-values <0.05 were indicated.


M2e5x VLP MPs and the combination VLP M2e5x VLP + Alhydrogel® + MPL-A® MPs elicited a significant increase in the expression of antigen-presenting molecules in dendritic cells compared to M2e5x VLP alone. Lastly, for preliminary evaluation of in vivo efficacy, the vaccine was administered in mice through the skin using an ablative laser. The M2e5x VLP + Alhydrogel® + MPL-A® MPs were shown to induce high levels of M2e-specific IgG antibodies. Further, a challenge with live influenza revealed heightened T-cell stimulation in immune organs of mice immunized with M2e5x VLP + Alhydrogel® + MPL-A® MPs. Hence, we utilized the advantages of both VLP and polymeric delivery platforms to enhance antigen immunogenicity and adaptive immunity in vivo.In this study, we demonstrate how encapsulating a conserved influenza ectodomain matrix-2 protein virus-like particle (M2e5x VLP) into a pre-crosslinked bovine serum albumin (BSA) polymeric matrix enhances in vitro antigen immunogenicity and in vivo efficacy. The spray-dried M2e5x VLP-loaded BSA microparticles (MPs) showed enhanced stimulation of antigen presenting cells (APCs), as confirmed through nitrite production and increased antigen–cell interactions seen in real time using live-cell imaging. Next, to further boost the immunogenicity of M2e5x VLP microparticles, M2e5x MPs were combined with Alhydrogel Influenza remains an ongoing public-health issue. Each year, influenza causes 3–5 million cases of severe illness and upwards of 650,000 deaths . AlthougThe ectodomain matrix-2 protein (M2e) of the influenza virus has been explored as a possible antigen for a universal influenza vaccine as it is highly conserved in influenza A strains. However, as M2e is only 23 amino acids, it has poor immunogenicity , liposomIn this study, we incorporated M2e5x VLP it into a polymeric microparticle delivery vehicle to further enhance its immunogenicity. Microparticles for vaccine delivery are advantageous as their size and shape mimic the size of bacterial pathogens, allowing for enhanced immune recognition and internalization by antigen-presenting cells (APCs) . EncapsuFormulation of a polymeric particulate vaccine can involve several techniques, including spray drying, emulsification, solvent evaporation, lyophilization, and coacervation . For forFor an effective vaccine, immunostimulation is important for inducing a robust immune response. Dendritic cells (DCs) are professional antigen-presentation cells and are the only type of phagocytes capable of stimulating primary and memory immune responses. Upon interaction with a pathogen, DCs takes up the pathogen through receptor-mediated endocytosis through specialized regions of the cell’s plasma membrane. Particulate and soluble antigens are internalized by DCs through phagocytosis and micropinocytosis, respectively . The cel®), is an oil emulsion shown to improve resistance against viral infections by modulating cytokine release by specifically driving a Th1 response [In addition to intrinsic immunopotentiating properties, particulate delivery systems can also be loaded with adjuvants to help boost the immune response towards pathogens, such as influenza, by focusing on stimulating specific toll-like receptor (TLR) agonists . Adjuvanresponse ,19.® + MPL-A® microparticulate vaccine delivered through skin.Lastly, we carried out a preliminary in vivo study to evaluate the efficacy of the vaccine microparticles delivered through the skin. We evaluated how administering vaccine microparticles through the skin using an ablative laser could potentially serve as a less-invasive, needle-free route for vaccine delivery. Laser microporation of skin to deliver vaccines has previously been shown to significantly improve antibody responses as the skin can serve as an antigen depot, allowing for slow diffusion of the vaccine into deeper layers of the skin and, therefore, extend antigen presentation . Therefo® or monophosphoryl lipid A (MPL-A®)  at a 5% w/v loading concentration.The antigen, M2e5x VLP, was provided by Dr. Sang-Moo Kang (Georgia State University). The construct consisted of heterologous tandem repeats of two human M2e (SLLTEVETPIRNEWGSRSN), swine M2e (SLLTEVETPTRSEWESRSS), type I avian M2e (SLLTEVETPTRNEWESRSS), and type II avian M2e (SLLTEVETLTRNGWGCRCS), as previously described ,22. In bM2e5x VLP BSA polymeric microparticles were characterized for their morphology, size, and charge. The morphology of the microparticles was visualized using scanning electron microscopy . Particle size and surface charge  were measured using a Malvern Zetasizer .4 cells/well. After the cells reached confluency, various treatments were introduced to the cells in 500 μL of complete DMEM  media: Cells Only (negative control), M-Vac , blank (unloaded) MPs, M2e5x VLP suspension, Alhydrogel® MPs, MPL-A® MPs, and M2e5x VLP MPs. For M2e5x VLP, 0.5 μg total M2e5x VLP suspension was added to the wells. For M2e5x VLP MP and adjuvant MP groups, approximately 10 μg MPs  was added to each well. The plates were incubated for 24 h after which the Griess’ Assay was performed to assess nitrite production. Post incubation, 500 μL of cell supernatant was removed from sample wells and centrifuged at 6000 rpm for 3 min to eliminate any cellular debris. Next, 50 μL of supernatant from each sample was spun down and added to a 96-well plate. Next, 50 μL of 1% sulphanilamide was added to the wells. The plates were then covered in foil and incubated at room temperature (RT) for 5 min in the dark. Next, 50 μL of 0.1% naphthylenediamine (NED) in 5% phosphoric acid was added to each well of the plate. The plates were then covered in foil and placed on a shaker for 5 min. The plates were then read at 540 nm using a BioTek Synergy plate reader .To assess the induction of nitric oxide by M2e5x VLP, M2e5x VLP MPs, and adjuvant particles in immune cells, dendritic cells  were plated in a 24-well plate at a density of 5 × 104 cells/well in a 24-well plate. The next day, the cells were incubated with 16 μg of M2e5x VLP MPs in 200 μL media for 1 h. The aforementioned particle concentration was found to be optimal for visualizing dendritic cell interactions with microparticles. The cells were recorded under a brightfield lens at 60× magnification using a BioTek Lionheart Live Cell Imager  for 1 h.Since antigen–cell interactions are important for uptake and presentation of antigens, a real-time live-cell imaging study was performed. The purpose of this study was to observe interactions between dendritic cells and an antigen presented in a microparticulate delivery vehicle. First, dendritic cells were seeded at 5 × 104 cells/well. After reaching confluency, the cells were pulsed with various treatments: Cells Only (negative control), M2e5x VLP (0.5 μg/well), M2e5x VLP MPs (10 μg/well M2e5x VLP MPs), and M2e5x VLP + Alhydrogel® + MPL-A® MPs . This ratio of M2e5x VLP MPs to adjuvant MPs (2:10:1) was selected as it would be the same ratio used for in vivo testing. The cells were incubated for 24 h. The cells were then gently scraped from the bottom of the wells and centrifuged for 10 min at 1200 rpm. Next, cells were resuspended in PBS and stained with either anti-mouse FITC-labeled MHC I and APC-labeled CD80 or anti-mouse PE-labeled MHC II and APC-labeled CD40. Cells were then analyzed using a flow cytometer .To assess in vitro antigen presentation, dendritic cells were plated in 24-well plates at a seeding density of 5 × 104 dendritic cells were plated per well. After cells reached confluency, various treatments were introduced to cells: 10 μL dimethyl sulfoxide , Cells Only (no treatment), and various concentrations of M2e5x VLP microparticles ranging from 31.25 to 1500 μg/mL. Each particle-containing well received 100 μL of particle suspension. For short-term cytotoxicity, the cells were treated for 24 h whereas for long-term cytotoxicity, the cells were treated for 96 h. After the incubation period, the cells were washed three times with PBS. Next, 10 μL of 0.5% w/v MTT reagent stock  was added to all wells. Thereafter, 90 μL of complete DMEM was added to each well. The plate was then incubated for 4 h to allow formation of the purple precipitate by metabolically active (live) cells. Next, media were removed and 100 μL of DMSO was added to each well. The plate was then covered in foil and placed on a shaker for 15 min to solubilize the precipitate. The plate was then read at 570 nm. The average absorbance of the cells-only group (untreated) was set as 100% cell viability. The following equation was used to calculate cell viability in other groups: average % cell viability = (absorbance of test group/absorbance of Cells Only) × 100.In a 96-well plate, 1 × 10n = 5): Naïve (negative control), Inactivated PR8 H1N1 (5 μg–positive control), and M2e5x VLP + Alhydrogel® + MPL-A® MPs (200 μg M2e5x VLP MPs + 1000 μg Alhydrogel® MPs + 100 μg of MPL-A® MPs). Two days prior to immunization, the dorsal sides of mice were shaved with a hair trimmer along with the addition of depilatory cream . For immunizations, the microparticles were suspended in PBS. All groups except Naïve received two applications of the Precise Laser Epidermal System (P.L.E.A.S.E.®) ablative laser. Laser ablation parameters consisted of a pulse duration of 125 microseconds, repetition rate of 200 Hz, 2 pulses/pore, pore diameter of 220 microns, pore density of 80 pores/cm2, and maximum pore depth of 2 mm. Next, the inactivated PR8 H1N1 or MP vaccine suspensions were pipetted onto newly formed micropores on the skin. Each mouse received 3 immunizations at 3-week intervals . Blood was collected at weeks 4, 7, and 10 for assessing serum IgG. In brief, whole blood was allowed to clot at room temperature for 30 min before being centrifuged at 1500 G for 10 min. The obtained serum was stored at −80 °C until use. Six weeks after the final booster, the mice were challenged intranasally with 50 μL of an 0.5 × LD50 dose of A/Philippines/2/82 (H3N2) to assess T-cell responses of vaccinated mice compared to naïve mice post challenge. The mice were monitored for 14 days post challenge after which they were sacrificed.For proof of concept to demonstrate how in vitro antigen presentation may translate to in vivo efficacy, 4–6-week-old Swiss Webster (CFW) mice  were immunized as follows (Serum IgG was assessed using ELISA. M2e was coated on 96-well plates at a concentration of 0.2 μg/well. Inactivated PR8 H1N1 was used as a coating antigen for the inactivated PR8 group (0.2 μg/well). The plates were incubated at 4 °C overnight. The next day, plates were washed three times with PBS + 0.1% Tween 20 (PBST) and blocked with 3% BSA (100 μL/well) for 2.5 h at RT. Serum samples were thawed on ice and serially diluted 10 times in PBST starting at 1:10 dilution. The plates were then washed, and the diluted samples (50 μL/well) were added to the plates and incubated at 37 °C for 1.5 h. The plates were then washed before the addition of 50 μL/well of horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG  at a dilution of 1:4000. The plates were then incubated at 37 °C for 1.5 h. Next, plates were washed and 50 μL of 3,3′,5,5′–Tetramethylbenzidine  was added to each well. The plates were incubated for 4 min at room temperature. The reaction was stopped by adding 50 μL/well of 0.3M sulfuric acid . Plate absorbance was measured at 450 nm using a plate reader.v/v DMSO and frozen at −80 °C. Single-cell suspensions of splenocytes were made by passing spleens through a 40 μm cell strainer. Lysis of red blood cells (RBCs) was carried out by adding ammonium chloride potassium (ACK) lysis buffer for 3 min. Cells were then washed and pelleted at 1200 rpm. Next, the cells were resuspended in freezing media and stored at −80 °C until analysis. For determining expression of CD4 and CD8 in lymphocytes and splenocytes, the cell suspensions were first thawed on ice and resuspended in PBS. Cells were then stimulated with 5 μg/mL IL-2 overnight and then labeled with anti-mouse FITC-labeled CD4 and APC-labeled CD8 antibodies. After 1 h incubation on ice, the cells were washed 3x and analyzed using flow cytometry.After animals were challenged and sacrificed, lymph nodes  and spleens were harvested. Single-cell suspensions of lymph nodes were prepared by passing organs through a 40 μm cell strainer. The cells were then centrifuged at 1200 rpm and resuspended in DMEM containing 70% fetal bovine serum (FBS) and 5% p-value < 0.05 was considered to be statistically significant.Data analyses were performed on GraphPad Prism Version 9.2. and expressed as mean values with a standard deviation (SD). For multiple comparisons, one-way analysis of variance (ANOVA) was performed with Tukey’s test for post hoc analysis. A The yield of M2e VLP-loaded BSA microparticles after spray drying was 92.4%. A Büchi Mini Spray Dryer B-290 setup is shown in p < 0.01).The production of nitric oxide by immune cells is one indicator of innate immunity, which can be a precursor for stimulating an immune response. For the Griess’ assay, the nitrite ions react in the Griess diazotization reaction to form a diazonium salt, which then reacts with N-(1-naphthyl)ethylenediamine in an azo coupling reaction, forming a pink-red azo dyenitric oxide that is oxidized to nitrite, which can be measured at an absorbance of 540 nm. Nitrite results are shown in Live-cell imaging at 60× magnification of antigen uptake by dendritic cells was visualized at various time points: 4, 25, and 45 min, as shown in p < 0.01) and M2e5x VLP + Alhydrogel® + MPL-A® MPs (**** p < 0.0001) compared to M2e5x VLP. CD80 expression in DC’s is significantly higher in M2e5x VLP + Alhydrogel® + MPL-A® MPs (*** p < 0.001) compared to M2e5x VLP. Expression of MHC II in M2e5x VLP MPs (** p < 0.01) and M2e5x VLP + Alhydrogel® + MPL-A® MPs (**** p < 0.001) is also significantly higher than M2e5x VLP. Lastly, for CD40 expression, M2e5x VLP MPs (* p < 0.05) and M2e5x VLP + Alhydrogel® + MPL-A® MPs (**** p < 0.001) are both statistically higher than M2e5x VLP. In conclusion, M2e5x VLP encapsulated in BSA microparticles (M2e5x VLP MPs) resulted in a significant increase in antigen presentation in MHC I, MHC II, and CD40 compared to M2e5x VLP alone. Additionally, the incorporation of adjuvants further improves the antigen presentation of the M2e5x VLP MPs as M2e VLP + Alhydrogel® + MPL-A® MPs demonstrated significantly higher expressions of MHC I, CD80, MHC II, and CD40 compared to M2e5x VLP alone.For antigen presentation, the percent of DCs expressing MHC I, CD80, MHC II, or CD40 is shown in p < 0.001) and 1500 μg/mL (* p < 0.05) were statistically different from Cells Only. The 1000 μg/mL concentration did not result in a significant reduction in cell viability compared to Cells Only. After 96 h exposure to M2e5x MPs, cell viability was concentration dependent, with higher MP concentrations demonstrating decreased cell viability. Cell viability for all M2e5x MP concentrations at 96 h was statistically lower than Cells Only. However, cell viabilities for all groups remained above 68%.Cell toxicity of M2e5x VLP MPs is shown in ® ablative laser and microporated skin after laser treatment is also shown in ® + MPL-A® MPs produced significant levels of M2e-specific antibodies compared to Naive. Inactivated PR8 H1N1-immunized mice (positive control) produced significant levels of PR8 H1N1-specific IgG. Hence, both Inactivated PR8 H1N1 and M2e5x VLP + Alhydrogel® + MPL-A® MPs demonstrated significantly higher IgG compared to the naïve group (**** p < 0.0001) at week 4 (post-first boost) and week 7 (post-second boost), remaining elevated through week 10.The dosing regimen, blood collection, and challenge schedule are shown in ® + MPL-A® BSA MPs showed heightened expression of CD4 and CD8 in T cells in spleen and lymph nodes compared to Naïve, which resembled the T-cell response to influenza infection in non-immunized mice. In lymph nodes, Inactivated PR8 H1N1 (** p < 0.01) and M2e5x VLP + Alhydrogel® + MPL-A® BSA MPs (** p < 0.01) showed significantly higher percentages of CD4+ cells compared to Naïve. For CD8 expression, similarly, both Inactivated PR8 H1N1 (* p < 0.05) and M2e5x VLP + Alhydrogel® + MPL-A® BSA MPs (** p < 0.01) showed significantly higher percentages of CD8+ T cells in lymph nodes compared to Naive. In splenocyte populations, Inactivated PR8 H1N1 (** p < 0.01) and M2e5x VLP + Alhydrogel® + MPL-A® BSA MPs (** p < 0.01) also exhibited significantly higher percentages of CD4+ cells compared to Naïve. Lastly, for CD8+ T cells in the spleen, Inactivated PR8 H1N1 (* p < 0.05) and M2e5x VLP + Alhydrogel® + MPL-A® BSA MPs (** p < 0.01) showed significantly higher percentages of cell expressing CD8 compared to Naïve.The challenge was not lethal for all mice, with no significant weight loss observed. CD4 and CD8 T-cell responses in lymph nodes  and splenocytes are shown in M2e was selected as the vaccine antigen as it is highly conserved in influenza A strains. The production of M2e-specific antibodies, although non-neutralizing, can confer cross-protection against various influenza strains by M2e antibody-dependent cell cytotoxicity (ADCC) . ResultaDendritic cell interactions with M2e5x VLP MPs were further assessed through real-time live-cell imaging as seen through dendrite rearrangements and dendritic pull of microparticle clusters. The live-cell imaging study confirmed that micron-sized particles can visibly enhance activity, interactions, and uptake by the dendritic cell body. This affinity is likely due to the size and irregular shape of the microparticles, as this high dendrite activity and reorganization was not seen in cells pulsed with M2e5x VLP alone (not shown). Additionally, the microparticle polymer itself, BSA, has been shown to have self-adjuvanting properties ,27, whic+ T cells. [Past studies have shown that the use of erodible microparticles containing antigen can act as an antigen depot upon contact with APCs, resulting in both intracellular and extracellular release of the antigen that prolongs antigen availability . The delT cells. . TherefoT cells. ,30,31,32® + MPL-A® MPs. ELISA results showed that immunizations with M2e5x VLP + Alhydrogel® + MPL-A® MPs were able to induce significant levels of M2e-specific antibodies. A similar trend was seen in CD4+ and CD8+ T-cell expression in lymphocyte and splenocyte populations of immunized mice post challenge. These preliminary results indicate that the vaccine microparticles administered through laser-microporated skin have the potential to elicit a strong immune response in vivo. This robust immune response is likely linked to large populations of Langerhans cells and dermal dendritic cells in the skin that can take up, process, and present the antigen to induce an immune response [As a proof of concept to explore if enhanced in vitro antigen presentation may translate to in vivo efficacy, mice were immunized through the skin with both an inactivated PR8 H1N1 (positive control) and the proposed MP vaccine consisting of M2e5x VLP + Alhydrogelresponse . Additioresponse . Thereforesponse .Future studies will address limitations of this current study pertaining to antibody and cellular responses in mice. Follow-up studies will address the role of antigen- and adjuvant-loaded polymeric microparticles on Th1/Th2-skewed, cytokine, and M2e-specific antibody/T-cell responses in M2e VLP MP groups compared to M2e VLP alone. Additionally, cross-subtype protection and potential immune responses towards the protein microparticles will also be assessed.® + MPL-A® MPs demonstrated enhanced in vitro antigen presentation and an increase in IgG (M2e-specific) and CD4+/CD8+ T cells in immunized mice. Hence, the incorporation of M2e5x VLP into an MP matrix with adjuvants and delivered non-invasively using laser ablation on the skin shows promise for the future development of a universal flu vaccine, capable of eliciting both innate and adaptive immunity against influenza.The M2e5x VLP formulated into polymeric microparticles and combined with adjuvants was shown to be a potential influenza vaccine candidate. The single-step spray-dried M2e5x VLP MP formulation was characterized for its properties, including shape, size, and charge. Furthermore, the M2e5x VLP MPs elicited significant nitrite production in dendritic cells. Furthermore, live-cell imaging demonstrated dendritic cells exhibiting a high affinity towards M2e VLP MPs. The final vaccine formulation consisting of M2e5x VLP + Alhydrogel


In their GTP-bound (active) form, Rab proteins interact with effector proteins that control downstream signaling. One such Rab15 effector is Rep15, which is known to have a role in receptor recycling from the endocytic recycling compartment but otherwise remains poorly characterized. Here, we report the characterization of the Rep15:Rab15 interaction and identification of Rab3 paralogs and Rab34 as Rep15 interacting partners from a yeast two-hybrid assay. Biochemical validation of the interactions is presented and crystal structures of the Rep15:Rab3B and Rep15:Rab3C complexes provide additional mechanistic insight. We find that Rep15 adopts a globular structure that is distinct from other reported Rab15, Rab3 and Rab34 effectors. Structure-based mutagenesis experiments explain the Rep15:Rab interaction specificity. Rep15 depletion in U138MG glioblastoma cells impairs cell proliferation, cell migration and receptor recycling, underscoring the need for further clarification of the role of Rep15 in cancer. Rep15 is an effector that interacts with the GTPase Rab15. Here, the authors show that Rep15 also interacts with Rab3 paralogs and Rab34, present crystal structures of Rep15:Rab complexes and find that Rep15 depletion in glioblastoma cells decreases proliferation and mobility. Rab GTPases have defined membrane locations and mediate the exchange of cargos between different organelles via selective binding and recruiting of distinct Rab interacting proteins2. Rab GTPases are the largest branch of the small GTPase family and more than 70 Rab family members have been reported so far. Similar to other small GTPases, Rab GTPases act as molecular switches. In the GTP-bound form, Rab is in an active state (on) and GDP-bound Rab is inactive (off)1. Two regulatory proteins tightly control the cycling between the on and off states. These are guanine nucleotide exchange factors (GEFs) that catalyze exchange of GDP by GTP and GTPase activating proteins (GAPs) that deactivate Rab by increasing the rate of GTP hydrolysis5. GTP-bound Rabs interact with various downstream effector molecules and Rab:effector complexes spatiotemporally control the distinct steps of vesicular trafficking1. Rab:effector recognition provides functional specificity and determines the various stages of trafficking. Most Rabs can interact with more than one effector molecule and similarly, effectors are also capable of interacting with phylogenetically similar Rabs8. Dysfunction of Rabs or their regulators or effector proteins contributes to various disease states11.Vesicular trafficking involves a precisely controlled flow of transport vesicles between the endocytic and secretory/exocytic pathways and this flow is tightly controlled by Rab GTPases13. In CHO cells, Rab15 co-localizes with Rab 4 and 5 on early endosomes/sorting endosomes (SE) and also with Rab11 on the endocytic recycling compartment (ERC)14, suggesting that it has distinct roles in the different endocytic compartments which can be achieved by recruiting different sets of effector molecules. In previous work, we have shown that Rab15 interacts with a plethora of bMERB family member effectors such as MICAL1, MICAL3, MICAL-cL, EHBP1 and EHBP1L116. However, additional studies are required to elucidate the functional significance of the Rab15:bMERB interaction. Apart from bMERB family members, Rep15 is another Rab15 effector molecule that regulates transferrin receptor trafficking. Recently multi-omics analysis data suggested REP15 as a novel colorectal cancer-specific driving gene17. Rep15 interacts with Rab15 at the ERC, and overexpression and depletion of Rep15 by si RNA attenuates the recycling of internalized transferrin receptor recycling via ERC without affecting receptor trafficking through sorting endosomes18. Rep15 is one of the least well-characterized Rab effectors; hence there remains a large gap in understanding the binding mode of the Rep15:Rab15 interaction and it also raises the question as to whether Rep15 only interacts with Rab15 or also has other uncharacterized interaction partners.Rab15 is enriched explicitly in the brain and its distinct isoforms are expressed in neuroblastoma cells19. The mammalian Rab 3 subfamily is comprised of 4 paralogs . Rab3 proteins are associated with secretory vesicles and have a role in the secretion of hormones and neurotransmitters20. Active Rab3 is localized to secretory vesicles and synaptic vesicles and dissociates upon depolarization of the nerve terminal. Active Rab3 has been shown to interact with various effector proteins, including Rabphilin3A21, Rabin3A22, RIM23, Noc224, synapsin25, Myo5a26, Slp4-a and NMHC IIA27. Single/double knockout mice of Rab3 paralogs are viable, whereas a triple knockout was lethal only if Rab3A was deleted. Knockout mice lacking all four mammalian RAB3 paralogs (RAB3A–D) show perinatal lethality, however, they show no changes in synaptic organization and only a minor change in neurotransmitter release28. This might be due to the presence of highly expressed Rab27A/B on the synaptic vesicles, since Rab3 and Rab27 share some common effector molecules29. Besides having a role in vesicular transport and early development, Rab 3 family members are also implicated in several cancers30, but effector molecules involved are still to be identified.Rab3 family members are also brain and neuro-endocrinal enriched GTPases and are implicated in the secretory pathwayHere, we describe the mechanistic basis of Rep15:Rab interactions and show that Rep15 forms a stable complex with Rab15 with 1:1 stoichiometry. Using the yeast two-hybrid approach, we have identified Rab3 paralogs and Rab34 as novel Rep15 interaction partners and further validated the interaction by co-localization and biochemical experiments. To decipher the binding mode, we have determined the structure of Rep15:Rab3B and Rep15:Rab3C complexes, respectively. The structures show that Rep15 is a globular Rab effector. Detailed biochemical studies reveal that Rep15 is selective for Rab15, Rab3 paralogs and Rab34 and does not bind to other evolutionarily similar Rab GTPases. Mutational analyses of both Rep15 and Rab3A reveal the molecular basis for Rab selectivity. Rep15 knockout experiments in U138MG glioblastoma cells show that Rep15 depletion leads to a decrease in cell proliferation and cell migration and also influences transferrin recycling.et al., have shown that the Rep15 is an effector molecule for Rab15 and co-localizes to endosomal membranes18. However, the biochemical characterization of this complex is not available. To understand the mechanism underlying Rep15:Rab15 complex formation, we purified full-length Rep15 and Rab15GppNHp and monitored their complex formation by analytical size exclusion chromatography (aSEC). Further, the interaction affinity was quantified by isothermal titration calorimetry (ITC) measurements  and in the present work, a naturally occurring N101D Rep15 variant (VAR_039548) was used  and dissociation (koff) rate constants were determined by transient kinetic experiments using a stopped-flow apparatus. For association rate constant measurements, 1 µM Rab15 loaded with fluorescent mant-GppNHp (2′/3′-O-(N-methyl-anthraniloyl)-guanosine-5’-[-imido]triphosphate) was mixed rapidly with increasing concentrations of Rep15. The association of mant-GppNHp Rab15 with the Rep15 leads to an increase in fluorescence polarization and the observed pseudo-first-order rate constant values (kobs) values were plotted against increasing concentration of Rep15. The association rate constant for Rep15 is 8.16 × 106 M−1 s−1 and the intercept on the y-axis yielded the dissociation rate constant of 4.73 s−1  for the complex was calculated as the ratio of koff/kon to have a value of 0.58 µM, in reasonable agreement with the value of 0.47 ± 0.12 µM obtained in ITC experiments. To measure the dissociation rate constant directly, the Rep15:mant-GppNHp Rab15 complex was rapidly mixed with a 25-fold excess of GppNHp Rab15. Dissociation of mant-GppNHp Rab15 was biphasic, with a faster dissociation rate constant of 2.28 s−1 (koff1) and a slower dissociation rate constant of 0.47 s−1 (koff2) was observed  complex and set up the crystallization screen; we also performed surface lysine methylation of the Rep15:Rab15 complex and also limited proteolysis of Rep15:Rab15GppNHp complex during crystallization; however, none of these trials yielded crystals.To help characterize the exact Rab15 binding region/domain, we generated a series of Rep15 deletion constructs based on the secondary structure prediction  screen using Rep15 against a constitutively active  human Rab library. We detected Rab3 family members and Rab34 as Rep15 interacting partners. However, we could not detect the Rep15:Rab15 interaction in our Y2H assay . To overcome the problem of heterogeneity, we expressed the dominant active Rab34Q111L_1-237aa that has reduced GTPase activity and purified the protein in the presence of GTP. Next, we monitored complex formation via aSEC  experiments and stable complex formation was detected with Rab3 paralogs and Rab34  and dissociation rate (koff) constants for the Rep15 and Rab3A interaction using a stopped-flow apparatus. 0.5 µM mant-GppNHp Rab3A was used against increasing concentrations of Rep15 and the association of Rep15 leads to a decrease in fluorescence intensity of mant-GppNHp Rab3A. The observed kobs values were plotted against increasing concentration of the Rep15 and an association rate constant of 9.72 × 106 M−1 s−1 was observed. A dissociation rate constant of 4.29 s−1 was obtained from the intercept on the Y-axis  and a slower dissociation rate constant of 0.82 s−1 (koff2) was observed  for the complex was calculated as the ratio of koff/kon, which is similar to the value obtained from ITC measurements.For biochemical characterization, we purified the full-length Rep15 and Rabs from b34 Fig. . To quaness Fig. . The affess Fig. . Furtherxis Fig. . Next, wved Fig. , and agaQ81L_19-217, Rep15 ΔN1:Rab3BQ81L_18-190 and Rep15 ΔN1:Rab34Q111L_1-237 complexes. Apart from human Rabs, we have also purified mouse Rab3A and bovine Rab3C and formed 1:1 complex with the human Rep15. All obtained complexes were used to perform the crystallization screen and finally, we obtained crystals for Rep15:Rab3CGppNHp_Q222H_10-227 (bovine), Rep15:Rab3BGppNHp and Rep15_ΔN1:Rab3BQ81L_18-190 complexes.Since we were not able to obtain crystals of the Rep15:Rab15 complex, we tried to crystallize the Rep15:Rab3 paralog complexes as well as the Rep15:Rab34 complex. To do this, we either mixed purified Rep15 and Rabs at 1:1 ratio or co-expressed and purified Rep15 ΔN1:Rab3AGppNHp_Q222H_10-227 (bovine) complex to a resolution of 2.52 Å, with the structure of Rab3A35 (PDB: 3RAB) as a search model using molecular replacement and Phenix autobuild36. A partial model was obtained that was manually built and refined to convergence. The structure is composed of one Rep15:Rab3CGppNHp_Q222H_10-227 complex per asymmetric unit. The crystallographic data collection and structure refinement statistics are provided in Supplementary Table To understand the molecular basis of the interaction between Rep15 and Rabs, we have determined the structure of the Rep15:Rab3C8, Rep15 is a single domain globular protein which presumably explains why the other three deletion Rep15 constructs were not soluble. No electron density was observed for the first 14 amino acids, the middle 117 to 130 amino acids and for the last residue. Rep15 is composed of 7α-helices and 7β-strands. The central core is made by the β-strands which are flanked by the α-helices . A DALI search37 against the PDB database revealed that the Rep15 fold shares some structural similarity with a Holliday junction resolving enzyme38  with an RMSD of 2.9 Å for 108 aligned cα residues  with an RMSD of 0.7 Å for 168 aligned Cα residues.The structure of Rep15:Rab3C reveals that, unlike most Rab effectors40 revealed that the Rep15:Rab3C complex has a total buried interface of 843.4 Å2. The binding interface has a central hydrophobic core that is supported by several polar interactions. The hydrophobic patch is composed of switch I (V63 and I65), interswitch residues (F67 and W84) and switch II residues  that interact with the hydrophobic side chains of L88Rep15, F89Rep15 from α3 and F151Rep15, L154Rep15 and I155Rep15 from α4. The side chain of L88Rep15 forms an extensive hydrophobic interaction with a conserved triad of aromatic amino acids , which are involved in all Rab:effector interactions41. The side chain of I94Rep15 interacts with V63 of switch I. The V135Rep15 hydrophobic side chain interacts with F67 while W140Rep15 hydrophobic side chain interacts with W84 and Y99. Several polar interactions were also observed in Rab3C and Rep15, including those between side chains D66-T82Rep15, D66-H84Rep15, R91-Q107Rep15, R91-D158Rep15, Y92-Q107Rep15, while the backbone of F67 forms a hydrogen bond with the side chain of H84Rep15 and the backbone of V63 and I65 form hydrogen bonds with the side chain of Q85Rep15. Sidechains of the interswitch residues K80 and Q82 form hydrogen bonds with the backbone of R134Rep15 , ß2 and β3 of Rep15. α3 of Rep15 interacts with the switch I and interswitch regions, while α4 and β3 interact with the switch II region of Rab3C. Analysis of the interface by PDBePISAp15 Fig. .GppNHp and Rep15 ΔN1:Rab3BQ81L_18-190 complex crystals, which diffracted to a resolution 2.75 Å and 2.8 Å, respectively and crystallized in space group C2 and P21 respectively. We solved the Rep15:Rab3B complex structure using Rep15:Rab3C (bovine) as a MR model  of switch I is replaced by a charged/polar residue in evolutionarily similar Rabs  of switch II residue is another non-conserved residue that interacts with the crucial Q107Rep15 and also interacts with I155Rep15. This tyrosine residue is replaced by phenylalanine in other Rab8 family members  and also in other exocytic Rabs . However, surprisingly in Rab34, this tyrosine residue is replaced by phenylalanine and a lysine residue replaces the neighboring arginine. Besides the changes in the switch II region, Rab34 has an extended N-terminus that may assist Rep15 interaction. To assess the significance of this tyrosine residue, we purified Rab27A_F81Y/Rab3A_Y84F/Rab34_Q111L_F114Y and checked the interaction with Rep15. Compared to wild-type Rab27A, the mutant F81Y Rab27A shows some complex formation with Rep15 in aSEC experiment and compared to wild type, the F81Y mutant shows more than an 8-fold increase in Rep15 binding affinity 42, ACAP2 43, the RUN domain of RUSC2 (interacts with Rab35 via 3 helices)43, MyoVb (binds to Rab11a via 3 helices and a loop)44, while the Rab5 binding interface of EEA1 is composed of a short β-hairpin and an α-helix45, whereas the OCRL Rab8 binding interface is made up of an Immunoglobulin-like β-strand domain and an α-helix46.Most Rab effectors bind to their cognate Rabs by 1 or 2 α-helices16, Rab3 paralogs47 and Rab3448 effector domains are made up of 1-3 α-helices, Rep15 is a globular effector with a 7α + 7β arrangement , the Rabphilin-3A:Rab3A complex47 (PDB: 1ZBD) structure has a larger binding interface (1426.5 Å2)  and Rab subfamily motifs (RabSF 1-4)49. RabF motifs cluster in and around switch regions whereas the RabSF1 motif overlaps with the RabCDR1, RabSF3 to RabCDR2 and RabSF4 to RabCDR3 region49  motif has phenylalanine whereas Rab3A has a tyrosine residue  which is also part of the binding interface  provides specificity in the case of Rab3 paralogs and Rab15.In contrast with previous studies suggesting that most of the Rab15ent Fig. . Unlike Å2) Fig. . The RabÅ2) Fig. . StructuÅ2) Fig. . RaphiliÅ2) Fig. . Similarn49 Fig. . The Rabn49 Fig.  and SlacFKCIA) and still interacts with Rep15. This can be explained by the fact that Rab34 is evolutionarily distant from the other Rep15 interacting Rabs and the Rab34 switch II region is quite different  is available  both showed a significant increase in proliferation compared to the KO cells  were significantly downregulated, while the expression of Rab3A remained unchanged , there was a slight trend in the upregulation of the expression of the Rab3B and Rab34, which was only significant in the case of Rab3B for the rescue with the WT construct  showed diminished migration over the 48 h period that was sufficient for the Cas9 control cells (UC) to completely close the wound   and their neighboring cells. This property, termed as anchorage independence of growth, correlates closely with tumorigenicityUC) Fig. . This phUC) Fig. .18, we performed transferrin (Tfn) uptake and recycling assays in the control, Rep15 KO and the two rescue cell lines . We could show that recycling of transferrin was significantly reduced in KO cells compared to control, indicated by significant retention of transferrin inside the cells . Overexpression of Rep15 in the WT cells also resulted in a significant decrease in the recycling of transferrin similar to the KO cells . A similar observation was made by Strick et al.18; this could be due to the titration of other partners involved in endocytic steps. Next, we repeated the above measurements in the KO and the two rescue cell lines to see the effect of the Rab binding deficient mutant (Q107A) in this process. The WT Rep15 (KOWT) could rescue transferrin recycling in the KO cells while overexpression of Q107A mutant (KOQ107A) did not affect this process  significantly reduced the recycling pathway while Q107A mutant (UCQ107A) did not , which shows minimal/no interaction with the Rab proteins. Since both the knockout and overexpression of Rep15 have a similar inhibitory effect on the recycling pathway the rescue experiments should be interpreted cautiously. Since we had mixed rescue phenotypes in different experiments, the physiological aspect of the effect of the Rab binding deficient (Q107A) mutant should be carefully validated in detail in future studies.Since Rep15 has been implicated in facilitating receptor recycling from the endocytic recycling compartment (ERC)ess Fig. . To furtess Fig. . The traess Fig. . FurtherMycobacterium bovis infected human macrophages showed that Rep15 is one of the genes overexpressed upon infection58. Recently, Rab3 paralogs have been implicated in various cancers56 and Rab34 is essential for ciliogenesis60. Taken together, this suggests that it would be important to study the role of Rep15 in cancer, ciliogenesis and Mycobacterium bovis-induced infection.Taken together, our data provide a detailed overview of the Rep15:Rab interaction. Here, we have identified Rab3 paralogs and Rab34 as novel Rep15 interaction partners and thoroughly characterized the Rep15:Rab interaction. We report the first Rep15 structure in a complex with Rab3B/C. Further, our mutagenesis experiments shed light on the origins of Rep15 selectivity towards specific Rabs. Additionally, we have demonstrated that loss of Rep15 results in decreased cell proliferation and migration of glioblastoma cell lines. Apart from having a role in receptor recycling, comparative proteomics studies of All the expression constructs used in this study were generated by standard cloning techniques, using Phusion polymerase, restriction digestion, and ligation by T4 DNA ligase. Point mutants were generated by quick-change site-directed mutagenesis, using Phusion polymerase. A detailed overview of all expression constructs employed in this study is presented in Supplementary Tables https://www.sigmaaldrich.com/DE/en/product/sigma/f7425, dilution 1:1000) and anti-alpha-Tubulin monoclonal (DM1A) mouse IgG1 T9026  were used as primary antibodies. IRDye® 800CW Goat anti-Rabbit IgG Secondary Antibody  and IRDye® 680RD Goat anti-Mouse IgG Secondary Antibody  were used as secondary antibodies.ANTI-FLAG® antibody produced in rabbit F7425  constructs were transformed into the yeast strain Y187  and transformants were selected on synthetic defined medium lacking tryptophan (SD-W). The pGADT7–Rep15 constructs were transformed in the yeast strain L40ΔGal4  and transformants were selected on synthetic defined medium lacking leucine (SD-L). For the mating experiments, 50 µL of bait and prey culture were added to 100 µL YPDA (Yeast peptone dextrose adenine) media in a 96-well plate (Sarstedt) and incubated 20-22 h at 30 °C with 180 r.p.m. Cells were resuspended and 5 µL were spotted on SD-LW (lacking leucine and tryptophan) plates as mating control, SD-LWH  and SD-LWH + 2.5 mM 3-Aminotriazol , an inhibitor of the histidine biosynthesis. Plates were incubated at 30 °C for 3–4 days and the strength of interaction was estimated from the comparative growth of yeast cells on SD-LWH or SD-LWH + 3′-AT plates.Yeast two-hybrid assay was performed, as described previouslyQ81L, Rab3BQ81L, Rab3CQ89L and Rab3DQ81L) were cloned into the pmCherry (C1) vector between XhoI and BamHI sites by conventional PCR using Y2H active Rab constructs as a template. The Rep15 was cloned into the pEGFP (N1) vector between XhoI and SalI sites and all constructs were verified by DNA sequencing. A detailed overview of constructs is presented in Supplementary Table 2. Cells were grown on a coverslip in 6 well plates until they reached 60–70% confluency and transiently transfected using linear polyethylenimine, MW 25000 . Expression was checked 16-24 h post transfection and cells were fixed with 3.7% paraformaldehyde in PBS for 15 min at room temperature. After washing with PBS, coverslips were mounted on glass slides with SlowFade Gold antifade reagent (Invitrogen). Images were taken by an EVOS fl fluorescence microscope equipped with 60X/1.42 Plan Apochromat oil immersion objective. Images were assembled with Adobe Illustrator.The full-length human mCherry-tagged active Rab constructs -RIL (Agilent Technologies) in lysogeny broth (LB) media supplemented with proper antibiotics and cells were lysed into a buffer containing 50 mM Hepes pH 8.0, 500 mM NaCl/LiCl, 1 mM MgCl2, 10 µM GDP, 2 mM βME (2-Mercaptoethanol), 1 mM PMSF (Phenylmethylsulfonyl fluoride) and subsequently disrupted by passing through a fluidizer (Microfluidic). Lysates were cleared by centrifugation at 75,600 g for 30 min and the supernatant was loaded onto Ni Sepharose  column. The bound proteins were eluted with an imidazole gradient (lysis buffer supplemented with 500 mM Imidazole pH 8.0). The His6-tag/His6-tag-MBP was cleaved by Tobacco Etch Virus (TEV)-protease during overnight dialysis at 4 °C in a buffer  and a second Ni2+-affinity purification was performed to remove the TEV protease and His6-tag/His6-tag-MBP. Rabs were preparatively loaded with GppNHp (Guanosine-5′-[β-γ-Imido]-triphosphate) or mant-GppNHp (2′/3′-O-N-Methyl-anthraniloyl)-guanosine-5′-[β-γ-imido]-triphosphate) or mant-dGppNHp (3′-O-(N-Methyl-anthraniloyl)−2′-deoxyguanosine-5’-[β-γ-imido]-triphosphate) and the reaction was performed, as described previously15. Further purity was achieved by gel filtration  . Residual MBP is removed by the amylose column.Rab GTPases were expressed and purified as described previously® Ultra-15 10 K NMWL centrifugal filter devices (Millipore). Nucleotide exchange efficiency was quantified by C18 reversed-phase column  with HPLC in 50 mM potassium phosphate buffer pH 6.6, 10 mM tetrabutylammonium bromide and 12% acetonitrile (v/v) and for the mant-GppNHp/mant-dGppNHp exchange the running buffer contains 25% acetonitrile (v/v). Protein samples were heat precipitated at 95 °C for 5 min and centrifuged at 15700 g for 10 min and loaded  on the column. Peaks were integrated and to determine the nucleotide retention times; a nucleotide standard run was performed.Rab34 is loaded with GTP, the reaction was performed in presence of 10x EDTA and 50x GTP and reaction was performed at RT for 1 hr and a further 2 hr on ice, and buffer was exchanged on AmiconE. coli BL21 Codon-Plus(DE3)-RIL cells in LB media supplemented with proper antibiotics and cells were grown at 37 °C until the optical density at 600 nm reached 0.8–1.0. Cells were stored at 4 °C for 20 min then 0.5 mM IPTG was added to induce expression and cells were allowed to grow at 20 °C for 14–16 h. Cells were pelleted down and stored at −80 °C until ready for purification. Cells were mechanically lysed by passing through a fluidizer (Microfluidics) in a buffer [Buffer A: 50 mM Tris pH 8.0, 500 mM NaCl, 2 mM βME, 1 mM PMSF and 1 mM CHAPS and lysates were cleared by centrifugation at 75,600 g at 4 °C for 30 min using a JA 25.50 rotor (Beckman-Coulter). Next, the proteins were purified by Ni2+-affinity chromatography and protein fractions were eluted with an imidazole gradient (Buffer B: buffer A supplemented with 500 mM imidazole pH 8.0). The His6-tag MBP was cleaved by Tobacco Etch Virus (TEV)-protease during overnight dialysis at 4 °C in a buffer  and a second Ni2+-affinity purification was performed to remove the TEV protease and His6-tag-MBP. Further purity was achieved by gel filtration  . Depending on purity, the protein was further purified by anion exchange chromatography . The purified protein was collected and concentrated and washed with final buffer  using Amicon® Ultra-15 10 K NMWL centrifugal filter devices; flash-frozen in liquid N2 and stored at −80 °C. Protein concentration was determined using by Bradford assay (Bio-Rad).Wild type and mutant His6-tagged MBP-Rep15 fusion proteins were recombinantly expressed in Q67L_1-176, Rep15 ΔN1:Rab3AQ81L_ΔCaaX and Rep15 ΔN1: Rab3BQ81L_18-190 complex co-purifications, Rabs (untagged) were cloned into pET30 vector between NdeI and XhoI restriction sites. His6 tagged-Rep15 and Rab were co-expressed in E. coli BL21 D3-RIL cells and cells were lysed in a buffer containing 50 mM Hepes pH 8.0, 150 mM NaCl, 1 mM MgCl2, 2 mM βME, 1 mM PMSF without CHAPS. The complexes were purified by Ni2+-affinity chromatography as described above. Final purity was achieved by gel filtration in the final storage buffer .For the Rep15 ΔN1:Rab152 and 2 mM DTE (Rab buffer). Samples were centrifuged for 15 min at 15700 g at 4 °C and 40 µl of the sample was injected into a Superdex 75 10/300 GL gel filtration column  pre-equilibrated with the buffer with a flow rate of 0.5 ml/min at room temperature and absorption at 280 nm was recorded.The Rep15: Rab complex formation was analyzed by analytical size exclusion chromatography (aSEC). 110 µM of Rep15 and 121 µM of Rab protein (Effector: Rab stoichiometry of 1: 1.1) were mixed in a buffer containing 20 mM Hepes 8.0, 50 mM NaCl, 1 mM MgCl2 and 2 mM DTE) using a SX-20 stopped-flow apparatus (Applied Photophysics) at 25 °C. The experiments were performed using the signal from the methylanthraniloyl group of mant-GppNHp/mant-dGppNHp, excited with a 365 nm and emission was detected through a 420 nm cutoff filter. Change in the fluorescence polarization of mant signal of 1 µM Rab15mant-GppNHp (post-mix) was observed with an increasing amount of Rep15. All stopped-flow results that were analyzed are averages of 6–8 individual traces. Single/double exponential functions were fit using the Origin9 software (OriginLab).Rep15: mant-GppNHp Rab15 kinetic measurements were performed in Rab buffer  was observed with an increasing amount of Rep15 .For the Rep15: mant-GppNHp kinetics measurements, a change in the fluorescence intensity of mant signal of 0.5 µM Rab3A2 and 1 mM Tris (2-carboxymethyl) phosphine (TCEP). To remove buffer mismatch, proteins were dialyzed overnight in the buffer and samples were centrifuged at 15700 g for 30 min at 4 °C. Protein concentration was determined by the Bradford assay (Bio-Rad). 500/600 µM of GppNHp/GTP Rab was titrated into the cell containing 50/60 µM Rep15. For the control experiments, the buffer was titrated into the cell containing the Rep15 and in the second control experiment, the Rab was titrated against the buffer. The binding isotherms were integrated, corrected for the offset and the data were fitted to a one-site-binding model using Origin 7.0 . The reported ITC results are representative of at least three independent measurements. Thermodynamic parameters are summarized in Supplementary Table Rep15-Rab interaction measurements were conducted by isothermal titration calorimetry (ITC) using an ITC200 or PEAQ-ITC microcalorimeter . A typical ITC experiment is consisted of 19 injections, with the first injection of 0.5 µL followed by 18 injections of 2 µL of Rab in a buffer containing 20 mM Hepes 8.0, 50 mM NaCl, 1 mM MgClGppNHp_Q222H_10-227 (bovine) (150 µM of 1:1 complex) was crystallized in 0.1 M MES pH 6.0 and 10% (w/v) PEG 6000. The complex of human Rep15:Rab3BGppNHp (300 µM of 1:1 complex) was crystallized in 0.1 M Bis-Tris pH 7.5, 0.2 M NaI and 20% (w/v) PEG 3350. Further, we crystallized co-purified human Rep15 ΔN1:Rab3BQ81L_18-190 (22 mg/ml) in 0.1 M Bis-Tris pH 8.5, 0.2 M KNa-tartrate and 20% (w/v) PEG 3350. The crystals were briefly soaked in a reservoir solution supplemented with 10% glycerol and flash-cooled in liquid nitrogen. Diffraction data were collected at 100 K on beamline X10SA at the Swiss Light Source . A native data set for the Rep15: Rab3CGppNHp_Q222H_10-227 (bovine) complex crystal was collected at a wavelength of 0.977930 Å whereas a data set from the Rep15:Rab3BGppNHp complex at a wavelength of 0.999961 Å. A native data set was collected for the Rep15ΔN1:Rab3BQ81L_18-190 complex at a wavelength of 0.977930 Å. Data were integrated and scaled with XDS62.For all protein complexes, initial crystallization condition screens were performed with the JCSG Core I–IV, Pact, and Protein Complex suites (Qiagen). The sitting-drop vapor diffusion method was used, with a reservoir volume of 70 μl and a drop volume of 0.1 μl protein  and 0.1 μl reservoir solution at 20 °C. The best conditions were then optimized using the sitting-drop vapor diffusion method varying drop sizes to obtain well diffracting crystals. The complex of Rep15 (human): Rab3CGppNHp_Q222H_10-227 (bovine) complex diffracted to a resolution of 2.52 Å  and a single copy of complex is present in the asymmetric unit of the crystal. The initial model for Rep15:Rab3CGppNHp_Q222H_10-227 (Bovine) complex was obtained by molecular replacement using PHASER63 with the structure of the Rab3A (PDB: 3RAB)35 as a search model. The partial model was completed with PHENIX AutoBuild36 and manual building in Coot64. The final models were refined to convergence with phenix.refine65. For the Rep15:Rab3BGppNHp complex, the crystal diffracted to a resolution of 2.75 Å  and for the Rep15ΔN1:Rab3BQ81L_18-190 complex, the crystal diffracted to a resolution of 2.8 Å  and two copies of the complex constitute the asymmetric unit. Rep15:Rab3CGppNHp_Q222H_10-227 (bovine) complex was used as a model for molecular replacement.The Crystal of Rep15:Rab3Chttp://www.pymol.org/).Data collection and refinement statistics are summarized in Supplementary Table TM) glioblastoma cell line was chosen as they show the maximum level of RNA as per Human Protein Atlas database. Of note, many different cell lines were checked for the expression of the Rep15 by real-time quantitative PCR analysis, they all showed very low levels of expression. U138MG cells showed slightly better expression among the tested cell lines, although the expression was very low in these cells also. Further, the cells were cultured in DMEM medium supplemented with 10% FBS, L-Glutamate, Na-Pyruvate and penicillin-streptavidin antibiotic. Two guide RNA (sg1 5′−ACTGACCACCTCACAGACGA−3′ and sg2 5′−TTAATAGGAGAGGATTGCCT 3′) were chosen to delete the majority of the coding region of the Rep15 gene and were cloned into lentiCRISPR V2 vector . The cells were transduced and selected with puromycin for a week before the single-cell dilutions were done in 96 well plates to generate single-cell clones. The genomic DNA was isolated from single-cell clones and PCR was done using primer pairs (FP 5′ - GAACCATTAACAGATGTGGCCT −3′ and RP 5′ - TGGAGGATATTCAAATCCAAGG −3′) flanking the two guide RNA to confirm the deletion of the coding region  for 3–4 weeks before the experiments were performed. As strong overexpression of Rep15 also led to inhibition of Rep15 mediated function such as receptor mediated recycling of the transferrin, we waited for the expression to decrease slightly in our mixed stable cell population before performing the rescue experiments. The overexpression of the WT and Q107A mutant was validated by western blot against the Flag-tag and tublin served as a loading control Fig. .66. For statistical analysis, the Student’s t-test (two-tailed) was applied.RNA was extracted using NucleoSpin RNA, Mini kit for RNA purification (MACHEREY-NAGEL Inc) and cDNA was synthesized using Verso cDNA Synthesis Kit (ThermoFisher Scientific), as per manufactures instructions. Real-time PCR amplification and analysis were performed using a LightCycler 480 with DyNAmo ColorFlash SYBR Green  and the primers used in the study are listed in Supplementary Table WT and KOQ107A) were seeded in a six-well plate format and they were trypsinized and counted on subsequent days (every day starting from day 2 till day 6). The results are from four independent biological repeats over four different passages.For cell proliferation measurements, 50,000 cells , in a 12-well plate. After the overnight incubation, the inserts were carefully removed and the cells were washed with PBS before exchanging the medium containing 2% FBS. A picture of the gap was taken immediately which served as a 0 h time point. Subsequently, a picture was taken at every 24 h time point and a gap between the two-cell fronts was taken as an estimate for the cell migration. The experiment was repeated six independent times with different passages.http://opencfu.sourceforge.net). The experiment was repeated six independent times with different passages.For anchorage, independent assay 10,000 cells were used per 35 mm dish. The bottom layer was made by pipetting 3 ml of 0.5% noble agar , while the top layer consists of 0.3% agar (0.33 ml of 3% of agar in water mixed with 2.67 ml of 20% FBS containing medium) with the required number of cells. After pipetting the cell suspension, the agar was left to solidify, which had a jelly-like consistency. To prevent further drying of the agar and cells it was made sure that the cell culture incubator was properly humidified and each petri dish was layered with a fresh 500 µl medium per week. A well-formed colony was visible in one month, which was stained by pipetting 200 µl of 5 mg/ml of iodonitrotetrazolium chloride solution. The number of the colonies in each dish was counted using open available software open CFU (The efficiency of receptor-mediated endocytosis in wild-type (WT), knockout (KO) and the rescue cells were examined by following the uptake of Alexa-488-conjugated human transferrin (ThermoFisher Scientific). The cells were seeded into 12 well plates with coverslips and incubated in a complete medium overnight. The next day, the cells were washed with serum-free medium three times and incubated with serum-free medium for 2 h. The medium was replaced with a fresh serum-free medium containing Alexa-488-conjugated human transferrin (20 µg/mL). For transferrin recycling, cells were exposed to transferrin for 30 min, after washing with a fresh serum-free medium three times, the culture was fed with a fresh medium for transferrin chase. Cells were fixed (3.7% PFA in PBS) at 2 h post washing and pictures were taken by a ZEISS Axio Observer fluorescence microscope. The intensity of the internalized transferrin was measured by ImageJ software and significance was calculated using the Student’s t-test (two-tailed). For the rescue experiment with transiently transfected GFP-tagged Rep15 constructs, Alexa-647-conjugated human transferrin (ThermoFisher Scientific) was used. The cells were seeded on the glass coverslip and the transferrin uptake and recycling experiment were performed exactly as described above after 48 h post- transfection. The experiment was repeated three times independently.67 was used to generate multiple sequence alignments. The protein interaction interfaces from the asymmetric unit were examined in detail using the PDBePISA server 40. DALI server was used for structural comparison37.Clustal OmegaFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Aberrant DNA methylation is one of the earliest hallmarks of cancer. DNMT1 is responsible for methylating newly replicated DNA, but the precise regulation of DNMT1 to ensure faithful DNA methylation remains poorly understood. A link between RNA and chromatin-associated proteins has recently emerged, and several studies have shown that DNMT1 can be regulated by a variety of RNAs. In this study, we have confirmed that human DNMT1 indeed interacts with multiple RNAs, including its own nuclear mRNA. Unexpectedly, we found that DNMT1 exhibits a strong and specific affinity for GU-rich RNAs that form a pUG-fold, a noncanonical G-quadruplex. We find that pUG-fold-capable RNAs inhibit DNMT1 activity by inhibiting binding of hemimethylated DNA, and we additionally provide evidence for multiple RNA binding modes with DNMT1. Together, our data indicate that a human chromatin-associated protein binds to and is regulated by pUG-fold RNA. Many chromatin-modifying enzymes that bind DNA and histones have also been found to bind RNA. This RNA binding has been proposed to play a role in both the recruitment and regulation of enzyme activity, but detailed mechanisms of RNA-mediated regulation remain poorly understood . ExampleThe DNA methyltransferases methylate cytosines within CpG dinucleotides—a major mechanism of epigenetic gene repression . CpG islCEBPA locus  which is down-regulated in colon cancer, was shown to interact with DNMT1 . Loss ofPA locus . DNMT1 iAdditionally, DNMT1 has been shown to be inhibited by binding to various miRNAs, including miR-155-5p . This RNIn this study, we investigate the sequence and structure specificity of DNMT1-RNA interactions and the effect of RNA binding on methyltransferase activity. We first use formaldehyde RNA immunoprecipitation, followed by next-generation sequencing (fRIP-seq) on endogenous DNMT1 in two different cell lines, and find that DNMT1 interacts with many RNAs, and surprisingly, its own mRNA. Upon further analysis of RNA sequence and structure preference of DNMT1, we see a high and specific affinity for a recently discovered RNA structure called a poly-(UG)-fold (pUG-fold) . We alsoSupplemental Fig. S1A,B). Additionally, we performed fRIP-seq with two different DNMT1-targeting antibodies to identify consistent RNA binding events. There was good overlap between the data sets from both antibody pulldowns in both cell types . DNMT1 bound about 3300 different RNAs in K562 cells and about 1200 in iPSCs ; this difference correlates with the higher level of DNMT1 in the K562 cell line as measured by western blot signal intensity . Most of the significantly enriched RNAs were mRNAs, and one of the most consistently enriched RNAs, regardless of cell line or antibody, was the DNMT1 mRNA .To dissect the binding preference of DNMT1 to various RNAs, we performed DNMT1 fRIP-seq in two different human cell lines, the leukemia cell line K562 and induced pluripotent stem cells (iPSCs) . However, the interaction between DNMT1 and its mRNA was biased to the fully spliced and processed mRNA as evidenced by peaks observed mainly over exonic regions . Because DNMT1 is a nuclear protein, its preference for binding to the fully spliced transcript was unexpected. To test if the observed interaction between DNMT1 and its mRNA could be due to crosslinking of the nascent DNMT1 peptide and the mRNA in the cytoplasm during translation, we performed the same fRIP-seq experiments after nuclear fractionation . We observed the same DNMT1–mRNA interaction in the nucleus, thereby supporting the hypothesis that this interaction is a nuclear binding event . In addition to binding to the fully spliced transcript, we noticed a preference for binding toward the 3′ end of the mRNA. Although there was a slight PCR amplification bias toward the 3′ end of the DNMT1 mRNA, it was not enough to explain the observed 3′-binding preference of DNMT1 .The highest percentage of DNMT1 fRIP-seq peaks was within introns and regulatory regions . These RNAs span the regions that show peaks in the fRIP-seq of Supplemental Fig. S3A,B). While all RNAs bound with similar affinities, RNA3 had the highest affinity (∼250 nM Kdapp) . In general, the affinity of DNMT1 for the various mRNA truncations is similar regardless of sequence composition or predicted amount of secondary structure, indicating that DNMT1 has a broad general affinity for RNA.To further test the affinity of DNMT1 to its own mRNA, we made six ∼200 nt RNA truncations by in vitro transcription (M Kdapp) B,D. Seve40 and undetectable binding to (A)40 (10 bound with somewhat higher affinity than (GA)20, which has the same base composition but cannot form G4s. The observed higher affinity for G4s is consistent with previous studies showing direct interactions of DNMT1 with both RNA and DNA G4s (20 (69 nM Kdapp) to (A)40 C,E. The  DNA G4s . Additio DNA G4s . InteresM Kdapp) C,E. IntrSupplemental Fig. S4A; Supplemental Table 1). All stem–loop constructs ran with much higher mobility on a native gel compared to (GU)20, indicating that (GU)20 does not in fact form a stem–loop structure under these conditions . This idea was further supported by the fact that while DNMT1 bound to all these stem–loop structures, it had very low affinity, Kdapp > 1 µM . In addition, a 22 nt stem–loop RNA (R5) that was previously shown to bind to DNMT1 (Kdapp) in our conditions .Since G and U can form wobble base pairs, it seemed possible that DNMT1 might be recognizing a stem–loop structure with G-U pairs in the stem and a hairpin loop comprised of GU repeats. We therefore designed hairpins with different sequence compositions and loop sizes, and we added GU repeats within the loops to tease apart potential binding preferences of the loop versus stem 20 ran with much faster mobility in K+ compared to Li+, indicating it is forming a K+-dependent compact structure 20 binding affinity was substantially reduced (eightfold) in Li+ compared to K+ (+) and unfolded GU repeats (Li+), with substantial preference for the folded form. Intriguingly, DNMT1 seems to exhibit a specific affinity for pUG-folds compared to canonical G4s, as indicated by the increased affinity for (GU)20 over RNAs of equal length that can form canonical G4s .12 and (GU)11 in both K+ and Li+. As predicted by the pUG-fold hypothesis, (GU)12 in K+ had a significantly higher affinity for DNMT1 than (GU)12 in Li+ or (GU)11 in either K+ or Li+ + or Li+ E. Import12 has significantly reduced affinity (fivefold) compared to (GU)20, indicating that one pUG-fold alone does not confer very high affinity for DNMT1 (30. As expected from the GU repeat length-dependent affinity observed in the previous experiment (30 (Kdapp = 13 nM) compared to (GU)20 . Interestingly, (GU)30 may be capable of forming two pUG-folds, or some other extended structure composed of G-quartets. To test if this is indeed the case, we repeated the same binding assay in Li+ and found a >50-fold reduction in affinity, arguing that the increased capacity to form pUG-folds or related structures indeed contributes greatly to DNMT1 binding affinity . To test if this GU repeat length–specific increase in binding affinity is specific to GU repeats, we performed the same experiment with (AC)20 and (AC)30. Notably, increased AC repeat content did not increase affinity, as seen with increasing GU repeats . Together, these results demonstrate that DNMT1 shows a high and specific affinity for pUG-fold RNA.Interestingly, (GU)or DNMT1 E. To detperiment E, DNMT1 12, (GU)20, and (GU)30. Reduced CD signal for both (GU)20 and (GU)30, as well as increased absorbance at 260 nm, indicated that both RNAs have single-stranded GU sequences that reduce the overall pUG-fold signal, compared to (GU)12, which had a very strong pUG-fold signal . All RNAs had identical spectra in LiCl, indicating that, regardless of length, each RNA forms a similar unstructured conformation in the absence of K+ ions. Furthermore, thermal melting analysis at 244 and 264 nm—the two wavelengths that are most indicative of the pUG-fold—demonstrated that all RNAs had similar two-step melting curves with the major melting transition around 55°C ; this is similar to the reported melting transition for a pUG-fold  and a thermal melting analysis with (GU)pUG-fold . Interesical G4s .20 or (A)40 in physiologically relevant KCl concentrations (100 mM). Interestingly, 1 µM (GU)20 mostly abolished activity while (A)40 had no effect at the same concentration (30 was stronger than that of (GU)20 , which is consistent with it having the highest binding affinity of all RNAs tested 10 but no G4-potential, mildly inhibited activity of DNMT1. Like (A)40, (C)40 also showed no inhibition of DNMT1 activity, even though it has a much higher affinity for DNMT1 compared to (A)40 ntration A. We nexs tested . (GU)12,hylation B. In conactivity B. These fold RNA B. (GA)20to (A)40 . Togethe20 and (A)40 to compete with a radiolabeled hemimethylated DNA substrate for binding. We saw strong competition for binding by (GU)20 with IC50 ∼ 100 nM . These results indicate that pUG-fold RNA inhibits DNMT1 activity by preventing its hemimethylated DNA substrate from binding. Whether inhibition is due to direct competition in the active site or due to an allosteric change remains to be determined.To further understand the inhibition of DNMT1 activity, we measured the ability of cold (GU)DNMT1 mRNA that we initially observed in the fRIP-seq results. We wondered if the bias in affinity toward the 3′ end of mRNA may be explained by high GU-repeat content. We analyzed the GU-content of the 3′-UTR of the DNMT1 mRNA and noticed that it is indeed GU-rich with UG/GU being the most frequently occurring dinucleotide (20.8% GU/UG content) . To test if DNMT1 binds specifically to its own 3′UTR, and if this interaction is GU-repeat dependent, we first performed an EMSA with the full-length in vitro transcribed 321 nt 3′UTR (20 (both 69 nM Kdapp), although the larger size of 3′UTR RNA may contribute to its affinity .Next, we sought to determine if the affinity of DNMT1 for GU-rich RNAs explains the binding to the nt 3′UTR A. The 3′affinity . Mutatioaffinity A. Furtheaffinity A. The liWe next tested if the WT 3′UTR or the GU-stretch mutant would influence DNMT1's methyltransferase activity. Neither RNA affected activity significantly in vitro B. The hiHere, we have uncovered two modes of RNA-binding by DNMT1: a high and specific affinity for pUG-fold RNA and a moderate affinity, seemingly promiscuous binding to many other types of RNA. The pUG-fold mode of binding inhibits DNA methyltransferase activity in an RNA concentration–dependent manner, while the more promiscuous RNA binding has little to no effect. Given the diversity of RNA sequences and structures bound by DNMT1, there may in fact be more than two modes of binding. The second mode of binding is consistent with previous studies showing that DNMT1 binds to multiple different RNAs of varying length and secondary structure with variable effects on DNMT1 enzymatic activity. . AdditioC. elegans by recruiting the RNA-dependent RNA polymerase (RdRP) RRF-1 to synthesize siRNAs (40 and (GU)12 (40 having no effect and (GU)12 being strongly inhibitory.pUG-folds have been shown to contribute to RNA silencing in e siRNAs . As there siRNAs . To our e siRNAs . Furtherd (GU)12 ] have veWhile noncanonical G4s have been characterized previously, especially in RNA aptamers, there is limited information regarding the affinity of RNA-binding proteins (RBPs) for canonical versus noncanonical G4s . There iSupplemental Fig. S2A). Furthermore, DNMT1 binds with high affinity to its own 3′UTR but does not inhibit methyltransferase activity in vitro. This suggests that the DNMT1–DNMT1 mRNA interaction could be regulatory in cells, for example, by influencing the binding of DNMT1 to its chromatin substrate or sequestering mature DNMT1 mRNA in the nucleus, preventing it from being transported to the cytoplasm for translation. This could represent an autoregulatory mechanism to control DNMT1 levels in the cell.In this study, we found that DNMT1 binds to its own mRNA in two human cell types and that this interaction seems to be specific to the fully spliced transcript in the nucleus. Binding to a spliced transcript was unexpected, because on average DNMT1 fRIP signals for introns were higher than for exons, as expected for a nuclear protein 20] 20] . It is ig and washed with 10 mL cold PBS. Cells were spun down and the supernatant was removed. Cells were fixed in 40 ml PBS + 0.1% formaldehyde at room temperature for 10 min. The formaldehyde solution was then quenched with 2 mL 2.5 M glycine (drops added slowly), followed by rotation at room temperature for 5 min. Cells were spun down at 500g for 5 min at 4°C. Supernatant was removed and cells were resuspended in 10 mL cold PBS + 1× Protease Inhibitor (Pierce Tablets). Cells were spun down for 5 min at 500g and supernatant was discarded. Pellets were resuspended in 10 mL ice-cold PBS + 1× PIC. Cells were spun down at 500g for 5 min and pellets were flash-frozen in liquid nitrogen and stored at −80°C or immediately used.Ten million K562 cells (ATCC: CCL-243) or iPSCs (WTC-11) were grown for each experiment. Cells were spun down for 5 min at 500Crosslinked cell pellets were thawed and resuspended in 1 mL cold fRIP lysis buffer . Samples were rotated at 4°C for 10 min and then sonicated at 4°C in a Bioruptor UCD-200 (Diagenode) on the high setting using a 30 sec on/30 sec off cycle for 15 min. Cells were spun down at 13,000 RPM for 10 min at 4°C. Pellets were discarded, and 1 mL of cold fRIP wash buffer  was added to 1 mL of supernatant to bring volume to 2 mL. Sample was filtered through a 0.45-micron filter into a new 2-mL tube and sample was split evenly between two new tubes (each with ∼5 million cells worth of lysate).Pierce Protein A/G Dynabeads (Thermo Scientific) were equilibrated with cold fRIP wash buffer , and 25 µL beads were added to each tube of lysate. Tubes were rotated at 4°C for 30 min. Beads were pulled down with a magnetic rack and supernatant was transferred to a new tube (precleared lysate).Two 25 µL aliquots of precleared lysate were taken here as input and western blot samples. Precleared lysates were flash-frozen in liquid nitrogen and stored at −80°C until further use or used directly for IP.One tube of precleared lysate was thawed for each IP condition. An amount of 6 µg of appropriate antibody was added to the lysate . Tubes were vortexed gently and then rotated at 4°C for 2 h. An amount of 50 µL of equilibrated protein A/G magnetic beads was added to each tube of lysate. Samples were incubated for 1 h at 4°C while rotating. Beads were pulled and supernatant discarded. Beads were washed four times in 1 mL fRIP wash buffer , each time rotating at 4°C for 10 min. Before removing the last wash, 100 µL of beads were transferred to a new tube. This 10% aliquot served as an IP sample for a western blot, and the remaining 90% was used for isolating RNA. Beads were pulled down for both the 900 and 100 µL samples and supernatant removed. Beads were either stored at −20°C or continued with for reverse crosslinking. The input and IP samples were thawed and 31 µL water was added to the input, and 56 µL water was used to resuspend the IP beads. To each sample, 33 µL 3× RCL buffer , 0.2 mg Proteinase K (Invitrogen) and 1 µL RNasin Plus RNase Inhibitor (Promega) were added. Samples were incubated at 42°C for 1 h and then at 55°C for 1 h. RNA was isolated via phenol/chloroform extraction followed by ethanol precipitation. Each pellet was resuspended in 15 µL nuclease-free water.Purified RNA from input and IP samples were reverse transcribed, PCR amplified and barcoded using the KAPA RNA HyperPrep Kit with RiboErase. The resulting cDNA libraries were paired-end sequenced on an Illumina NextSeq 500 at a depth of 20–40 million reads/sample. All whole-cell fRIP experiments had three replicates and the nuclear fRIP had four replicates.https://github.com/ljansson/dnmt1.git.Read mapping and QC of sequenced samples was handled by nf-core/chipseq v1.1.0 pipeline . Reads wProtocol was adapted from the Bönisch lab . Twenty g for 5 min at 4°C. Supernatant was removed and each nuclear pellet was washed once with 1 mL 1× FL buffer . Pellets were spun down at 1000g for 5 min at 4°C. Supernatant was removed and pellets were resuspended in 500 µL of 1× fRIP lysis buffer . fRIP was then performed as described above for whole-cell pellets. Nuclear fractionation was confirmed by western blot. Antibodies used for western blot include: DNMT1 (Abcam 19905), EZH2 , and β-actin (Sigma-Aldrich A5441).Cells were spun down at 1000Sf9 cells were used to generate baculovirus stocks using the Bac-to-Bac system (Life Technologies), according to the manufacturer's instructions. A baculovirus stock carrying the gene for DNMT1 was used to infect 4–12 L of Sf9 cells at a density of 2 million cells/mL in Sf-900 III SFM (Invitrogen cat. # 12658–027). Multiple 1 L cell cultures in 4 L flasks (VWR cat. # 32645–044) were incubated for 72 h at 27°C, 130 revolutions per minute (rpm). Cells were harvested by centrifugation for 20 min at 2000 relative centrifugal force (RCF)  at 4°C, then frozen in liquid nitrogen and stored at −80°C until protein purification.g between each wash. MBP-DNMT1 was eluted in low salt buffer + 10 mM maltose. Elution was concentrated and cleaved overnight with 1:50 w/w ratio PreScission Protease at 4°C. Following cleavage, DNMT1 was purified by ion-exchange chromatography on a HiTrap 5 ml Heparin HP column (Cytiva), and fractions were pooled and concentrated. The heparin-purified sample was then further purified by size-exclusion chromatography on a Sepharose 6 Increase 10/300 GL column (Cytiva). Fractions were pooled and concentrated and aliquots of pure DNMT1 were flash-frozen and stored at −80°C until use.A DNMT1 cell pellet was weighed and 50 mL of lysis buffer  was added to every 6 g of pellet. Pellet was gently resuspended, followed by incubation at 4°C while rotating for 15 min. Cells were spun down at 18,000 rpm for 30 min at 4°C. Pellet was discarded and supernatant was incubated with equilibrated amylose resin  (1 mL resin/2 g pellet) for 90 min at 4°C while rotating. Sample was then washed in batch with first 10 cv lysis buffer, 16 cv high salt buffer , 16 cv low salt buffer  with 5 min spins at 1000DNMT1 mRNA fragments from a full-length human DNMT1 cDNA plasmid (plasmid details available upon request). PCR products were purified using the E.Z.N.A. Cycle Pure Kit (Omega Bio-tek) and used as templates for T7 in vitro transcription. The in vitro transcription reaction was incubated overnight at room temperature (37°C for 3′-UTR constructs). The reaction then incubated with Turbo DNase for 15 min. The RNA was then isolated using phenol/chloroform extraction and ethanol precipitation followed by PAGE purification.PCR primers were designed to PCR amplify 32P-ATP in 1× PNK buffer and incubated for 1 h at 37°C. Radiolabeled RNAs were purified using QuickSpin columns (Roche), and radioactivity was measured by a scintillation counter. End-labeled RNA was folded by first heating to 95°C for 5 min followed by snap cooling on ice for 2 min, and then incubated for 30 min at 37°C in 1× refold buffer . Each binding reaction contained the indicated amount of DNMT1 plus 1000 cpm of end-labeled RNA in binding buffer . Reactions were incubated for 1 h at 30°C. Samples were run on a 1% agarose gel in 1× TBE and run for 90 min at 4°C at 66 V. The gel was dried and exposed overnight followed by imaging on a Typhoon FLA 9500 scanner (GE). All EMSAs were performed in triplicate. EMSAs were quantified using either ImageQuant or GelAnalyzer 19.1, and binding curves were generated using Prism 9. 40-mer RNAs were ordered from Dharmacon.In vitro transcribed RNAs were CIP-treated before end-labeling. An amount of 50 pmol of in vitro transcribed or synthetic RNA was incubated with 10 units T4 PNK (NEB) and 2 µL γ-2, 0.1 mM ZnCl2, 2.0 mM BME, 0.05% NP40, 5% v/v glycerol). Each binding reaction contained 0.5 µM DNMT1, 1000 cpm of end-labeled DNA and the indicated concentration of cold RNA in 1× binding buffer . DNA was directly diluted in 1× folding buffer before incubating with DNMT1 and cold RNA. Hemimethylated DNA was ordered preannealed from IDT: ([5′TA(5mC)GTATC(5mC)GTATC(5mC)GGTTA(5mC)GTATC(5mC)GAATC(5mC)GTAC(5mC)GT 3′/5′ ACGGTACGGATTCGGATACGTAACCGGATACGGATACGTA 3′]). CpG-flanking sequences were designed to promote ideal binding/activity by DNMT1 . All competition EMSAs were performed in triplicate. Binding competition assays were quantified using GelAnalyzer 19.1, and binding curves were generated using Prism 9. The amount of dissociated DNA was calculated by first subtracting the unbound DNA signal in the no RNA lane from all other lanes. Then, the fraction of bound DNA was calculated by dividing the signal from the bound band from total signal (bound plus unbound with smear in between both bands’ background subtracted).2, 0.1 mM ZnCl2, 2.0 mM BME, 0.05% NP40, 5% v/v glycerol, 0.1 mg/mL BSA [NEB], 0.1 mg/mL baker's yeast tRNA [Sigma R5636]) and 1.0 µM [3H] SAM . First, buffer, DNA, and [3H] SAM were combined in a master mix and aliquoted to appropriate tubes, followed by addition of indicated RNA. The reaction was then started by addition of protein. This order-of-addition was designed to ensure that the SAM, which is dissolved in acid, was neutralized with buffer before the protein was added. Reactions were incubated for 2.5 h at 37°C. Reactions were cooled to 4°C for 30 sec, quenched with 1.2 mM unlabeled SAM (NEB) and blotted onto Hybond-XL membrane. Membranes were allowed to air dry, then were batch washed (8 mL wash buffer per membrane) with 3× 50 mM KH2PO4, 1× 80% ethanol, 1× 100% ethanol. Membranes were allowed to air dry, then were soaked in ScintiSafe Econo 1 Scintillation Cocktail. A Beckman LS 6500 Scintillation Counter was used to measure 3H incorporation.Single point biochemistry assays were performed by incubating 0.25 µM DNMT1, 1.0 µM hemimethylated DNA (same as used for binding competition EMSAs), 1.0 µM folded RNA in 1× binding buffer  and various amounts of refolded RNA in 1× binding buffer. The same incubation, quenching, blotting, and washing protocols were followed as described above. Plots were generated using Prism 9.The inhibition patterns of various RNAs were determined by incubating 0.25 µM DNMT1, 1 µM hemimethylated DNA, 1 µM  was normalized by converting the absorbance values at 20°C to 1 (for 264 nm) or −1 (for 244 nm). Thermal melts were performed three independent times at 264 nm and twice at 244 nm with similar results each day. First derivative curves (dF/dT) were generated in Prism and curves were smoothed with a second order polynomial with 10 neighbors on each side.Thermal melting analysis was performed from 20°C–85°C at 244 and 264 nm at a rate of 1°C/min with a 20 nm step size. RNAs were prepared and folded as described above. The change in molar ellipticity [Δε at 37°C for 30 min. RNAs were loaded onto a 10% Native PAGE gel containing either 100 mM KCl or LiCl in 0.5× TBE. Samples were run at 80 V for 1 h in 0.5× TBE buffer plus 100 mM KCl/LiCl at room temperature. Gels were post-stained with SybrGold for 15 min and imaged by a FluorChem R Imager (ProteinSimple). Folding of DNMT1 mRNA in vitro transcription products was analyzed by loading onto a 6% PAGE gel without additional salt in gel and loading buffer.RNAs were heated at 95°C for 5 min and then snap cooled followed by folding in 1× refold buffer containing either KCl or LiCl (50 mM Tris-HCl pH 7.5, 100 mM KCl/LiCl, 2.5 mM MgClSupplemental material is available for this article.T.R.C. is a scientific advisor for Storm Therapeutics, Eikon Therapeutics, and Somalogic, Inc.