High-resolution Profiling of RNA-Protein Interaction in Living Cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE237019 | GEO | 2025/06/04
REPOSITORIES: GEO
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Project description:DNA-protein interactions regulate critical biological processes. Identifying proteins that bind to specific, functional genomic loci is essential for understanding the underlying regulatory mechanisms on a molecular level. Here, we describe a novel co-binding-mediated protein profiling (CMPP) strategy to investigate the interactome of DNA G-quadruplexes (G4s) in cellular chromatin. CMPP involves cell-permeable, functionalized G4-ligand probes that bind endogenous G4s and subsequently crosslink to co-binding G4-interacting proteins in situ. We show the robustness of CMPP on proximity labelling of a G4 binding protein in vitro. Employing this approach in live cells, we identify hundreds of putative G4-interacting proteins from various functional classes. Next, we observe high G4 binding affinity and selectivity for several G4 interactors in vitro and confirm direct G4 interactions for one of the top candidates in chromatin. Our studies provide a chemical approach to map protein interactions of specific nucleic acid features in living cells.
2021-06-28 | GSE165124 | GEO
Project description:The fetal-to-adult hemoglobin transition is clinically relevant as reactivation of fetal hemoglobin (HbF) significantly reduces morbidity and mortality associated with sickle cell disease (SCD) and b-thalassemia. Most studies on the developmental regulation of the globin genes, including genome-wide genetics screens, have focused on DNA binding proteins including BCL11A and ZBTB7A/LRF and their cofactors. Our understanding of RNA binding proteins (RBPs) in this process is much more limited. Two RBPs, LIN28B and IGF2BP1 are known post-transcriptional regulators of HbF production but a global view of RBPs is still lacking. Here, we carried out a CRISPR/Cas9-based screen targeting RBPs harboring RNA methyltransferase and/or RRM domains and identified RBM12 as a novel HbF suppressor. Depletion of RBM12 induced HbF expression and attenuated cell sickling in erythroid cells derived from SCD patients with minimal detrimental effects on cell maturation. Transcriptome and proteome profiling revealed that RBM12 functions independently of major known HbF regulators. Enhanced crosslinking and immunoprecipitation followed by high throughput sequencing (eCLIP-Seq) revealed strong preferential binding of RBM12 to 5’UTRs of transcripts, narrowing down the mechanism of RBM12 action. Notably, we pinpointed the first of five RRM domains as essential, and, in conjunction with a linker domain, as sufficient for RBM12-mediated HbF regulation. Our characterization of RBM12 as a negative regulator of HbF points to an additional regulatory layer of the fetal-to-adult hemoglobin switch and broadens the pool of potential therapeutic targets for SCD and b-thalassemia.
2022-06-14 | GSE200676 | GEO
Project description:The fetal-to-adult hemoglobin transition is clinically relevant as reactivation of fetal hemoglobin (HbF) significantly reduces morbidity and mortality associated with sickle cell disease (SCD) and b-thalassemia. Most studies on the developmental regulation of the globin genes, including genome-wide genetics screens, have focused on DNA binding proteins including BCL11A and ZBTB7A/LRF and their cofactors. Our understanding of RNA binding proteins (RBPs) in this process is much more limited. Two RBPs, LIN28B and IGF2BP1 are known post-transcriptional regulators of HbF production but a global view of RBPs is still lacking. Here, we carried out a CRISPR/Cas9-based screen targeting RBPs harboring RNA methyltransferase and/or RRM domains and identified RBM12 as a novel HbF suppressor. Depletion of RBM12 induced HbF expression and attenuated cell sickling in erythroid cells derived from SCD patients with minimal detrimental effects on cell maturation. Transcriptome and proteome profiling revealed that RBM12 functions independently of major known HbF regulators. Enhanced crosslinking and immunoprecipitation followed by high throughput sequencing (eCLIP-Seq) revealed strong preferential binding of RBM12 to 5’UTRs of transcripts, narrowing down the mechanism of RBM12 action. Notably, we pinpointed the first of five RRM domains as essential, and, in conjunction with a linker domain, as sufficient for RBM12-mediated HbF regulation. Our characterization of RBM12 as a negative regulator of HbF points to an additional regulatory layer of the fetal-to-adult hemoglobin switch and broadens the pool of potential therapeutic targets for SCD and b-thalassemia.
2022-06-14 | GSE200675 | GEO
Project description:Guanine (G)-rich nucleic acids can fold into G-quadruplex (G4) structures under permissive conditions. Although many RNAs contain sequences that fold into RNA G4s (rG4s) in vitro, their folding and functions in vivo are not well understood. Here, we showed that the folding of putative rG4s in human cells into rG4 structures was dynamically induced by stress. By using high-throughput dimethylsulfate probing, we identified hundreds of endogenous stress-induced rG4s. Our results demonstrated that stress-induced rG4s were enriched in mRNA 3′-untranslated regions and enhanced mRNA stability. Furthermore, stress-induced rG4 folding was readily reversible upon stress removal. In summary, our study revealed the dynamic regulation of rG4 folding in living human cells and suggests suggested that widespread rG4 motifs may have a global regulatory impact on mRNA stability and cellular stress response.
2022-12-15 | GSE200706 | GEO
Project description:A deeper understanding of the regulation of G protein-coupled receptors (GPCRs) and their associated downstream cascades will provide critical insights into how these pathways shape human physiology and disease, and could yield novel therapeutic targets. Here, we validate and characterize RNA-binding motif 12 (Rbm12) as a repressor of GPCR/cAMP signaling. We established Rbm12 CRISPR KO HEK293 cells using two independent gRNAs and human iPSC-derived neurons (iNeuron) depleted of Rbm12 via CRISPR interference. Then, we performed basal gene expression profiling (HEK293) and basal + beta-2-adrenergic receptor induced (1 hour of 1 uM Isoproterenol) conditions (neurons).
2023-08-02 | GSE219195 | GEO
Project description:RNA structures are of crucial importance for biological function and gene regulation. Guanine (G)-rich sequences in RNA can assemble to form RNA G-quadruplex (rG4) structures; specific rG4s have been shown to regulate gene expression, and are associated with human diseases. However, no transcriptome-wide method has been reported to map rG4s, limiting our understanding of the rG4 structure and its relationship with function and regulation. Here we introduce a transcriptome-wide rG4 profiling method, rG4-Seq, in which the rG4-mediated reverse transcriptase stalling is read out by next-generation sequencing at nucleotide resolution. We apply this method to human RNA and report the first global map of rG4 structures for any organism. Our analysis identifies over 3,000 rG4s, and increases to over 11,000 upon addition of a rG4 stabilizing ligand Pyridostatin (PDS). Notably, we discover that besides canonical rG4s, many rG4s are non-canonical with novel structural features such as long-loops, bulges, and 2-quartet. We also find that rG4 formation is associated with its stability, Cytosine (C)-content, and the stability of alternative RNA structures. Our data reveals that rG4s can be found in messenger RNAs (mRNAs) and long intergenic noncoding RNAs (lincRNAs). In mRNAs, rG4s are enriched in untranslated regions, and are significantly correlated with microRNA target sites and polyadenylation signals. Remarkably, we found that majority of our in vitro identified rG4s can change the in silico predicted RNA structure to uncover alternative RNA conformation.. Futhermore, the rG4s that have analogues in many ortholog genes across different species show a preferential association with RNA processing and stability. rG4-seq is a broadly applicable method that enables the RNA G4 structurome and its biological roles to be interrogated and can be readily applied in other organisms on a transcriptome-wide scale. 12 samples, 150 bp single-ended RNA-Seq libraries from cell extracts after performing RTS reaction in different buffers (Li, K and K+PDS rich buffer). Each of the 3 conditions has 4 libraries from independent biological replicates.
2016-08-10 | E-GEOD-77282 | biostudies-arrayexpress
Project description:Many biological processes are regulated by RNA-RNA interactions 1, nonetheless it remains formidable to analyze the entire RNA interactome. We developed a method, MARIO (MApping Rna-rna Interactions in vivO), to map protein-assisted RNA-RNA interactions in vivo. By circumventing the selection for a specific RNA-binding protein 2-5, our approach vastly expands the identifiable portion of the RNA interactome. Using this technology, we mapped the RNA interactome in mouse embryonic stem cells, which was composed of 46,780 RNA-RNA interactions. The RNA interactome was a scale-free network, with several lincRNAs and mRNAs emerging as hubs. We validated an interaction between two hubs, Malat1 and Slc2a3 using single molecule RNA fluorescence in situ hybridization. Base pairing was observed at the interaction sites of long RNAs, and was particularly strong in transposonRNA-mRNA and lincRNA-mRNA interactions. This reveals a new type of regulatory sequences acting in trans. Consistent with their hypothesized roles, the RNA interaction sites were more evolutionarily conserved than other regions of the transcripts. MARIO also provided new information on RNA structures, by simultaneously revealing the footprint of single stranded regions and the spatially proximal sites of each RNA. The unbiased mapping of the protein-assisted RNA interactome with minimum perturbation of cell physiology will greatly expand our capacity to investigate RNA functions. Three (3) ESC samples with different treatment (different digestion size and/or crosslinking method) and one (1) MEF sample were included to test our new approach for RNA-interactome mapping and the different samples were analyzed to show RNA interactome differences between them.
2016-06-23 | E-GEOD-61489 | biostudies-arrayexpress
Project description:Cancer cells rely on mitochondria for their bioenergetic machinery and macromolecule synthesis. The regulation of mitochondrial protein synthesis is central to mitochondrial function. This process primarily depends on the cytoplasmic translation of nuclear-encoded mitochondrial mRNAs whose protein products are imported into mitochondria. Despite the growing evidence that mitochondrial protein synthesis contributes to the onset and progression of cancer, and offers new opportunities for cancer therapy, knowledge of the underlying molecular mechanisms remains limited. Here, we show that RNA G-quadruplexes (RG4s) are localized to mitochondria and regulate mitochondrial function by modulating cytoplasmic mRNA translation of nuclear-encoded mitochondrial proteins . Our data support a model whereby RG4 stabilization induced by small molecule ligands or by depleting RG4-binding proteins alters nuclear-encoded mitochondrial mRNAs protein synthesis. Ultimately, this impairs mitochondrial functions, affecting energy metabolism and consequently cancer cell proliferation.
2025-02-17 | GSE239640 | GEO
Project description:Many biological processes are regulated by RNA-RNA interactions 1, nonetheless it remains formidable to analyze the entire RNA interactome. We developed a method, MARIO (MApping Rna-rna Interactions in vivO), to map protein-assisted RNA-RNA interactions in vivo. By circumventing the selection for a specific RNA-binding protein 2-5, our approach vastly expands the identifiable portion of the RNA interactome. Using this technology, we mapped the RNA interactome in mouse embryonic stem cells, which was composed of 46,780 RNA-RNA interactions. The RNA interactome was a scale-free network, with several lincRNAs and mRNAs emerging as hubs. We validated an interaction between two hubs, Malat1 and Slc2a3 using single molecule RNA fluorescence in situ hybridization. Base pairing was observed at the interaction sites of long RNAs, and was particularly strong in transposonRNA-mRNA and lincRNA-mRNA interactions. This reveals a new type of regulatory sequences acting in trans. Consistent with their hypothesized roles, the RNA interaction sites were more evolutionarily conserved than other regions of the transcripts. MARIO also provided new information on RNA structures, by simultaneously revealing the footprint of single stranded regions and the spatially proximal sites of each RNA. The unbiased mapping of the protein-assisted RNA interactome with minimum perturbation of cell physiology will greatly expand our capacity to investigate RNA functions. Three (3) ESC samples were treated with one (1) type of antisense oligonucleotides as is described in Kretz M. et al. (Nature. 2013 Jan 10;493(7431):231-5) to show the RNA interaction among specific RNAs and verify the results from MARIO
2016-06-23 | E-GEOD-65849 | biostudies-arrayexpress