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RBFOX2 alters splicing outcome in distinct binding modes with multiple protein partners.

ABSTRACT: RBFOX2 controls the splicing of a large number of transcripts implicated in cell differentiation and development. Parsing RNA-binding protein datasets, we uncover that RBFOX2 can interact with hnRNPC, hnRNPM and SRSF1 to regulate splicing of a broad range of splicing events using different sequence motifs and binding modes. Using immunoprecipitation, specific RBP knockdown, RNA-seq and splice-sensitive PCR, we show that RBFOX2 can target splice sites using three binding configurations: single, multiple or secondary modes. In the single binding mode RBFOX2 is recruited to its target splice sites through a single canonical binding motif, while in the multiple binding mode RBFOX2 binding sites include the adjacent binding of at least one other RNA binding protein partner. Finally, in the secondary binding mode RBFOX2 likely does not bind the RNA directly but is recruited to splice sites lacking its canonical binding motif through the binding of one of its protein partners. These dynamic modes bind distinct sets of transcripts at different positions and distances relative to alternative splice sites explaining the heterogeneity of RBFOX2 targets and splicing outcomes.

SUBMITTER: Zhou D

PROVIDER: S-EPMC8373071 | biostudies-literature |

REPOSITORIES: biostudies-literature

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RBFOX2 alter splicing outcome in distinct binding modes with multiple protein partners

Project description:RBFOX2 controls the splicing of a large number of transcripts implicated in cell differentiation and development. Parsing RNA-binding protein datasets, we uncover that RBFOX2 can interact with hnRNPC, hnRNPM and SRSF1 to regulate splicing of a broad range of splicing events using different sequence motifs and binding modes. Using immunoprecipitation, specific RBP knockdown, RNA-seq and splice-sensitive PCR, we show that RBFOX2 can target splice sites using three binding configurations: single, multiple or secondary modes. In the single binding mode RBFOX2 is recruited to its target splice sites through a single canonical binding motif, while in the multiple binding mode RBFOX2 binding sites include the adjacent binding of at least one other RNA binding protein partner. Finally, in the secondary binding mode RBFOX2 likely does not bind the RNA directly but is recruited to splice sites lacking its canonical binding motif through the binding of one of its protein partners. These dynamic modes bind distinct sets of transcripts at different positions and distances relative to alternative splice sites explaining the heterogeneity of RBFOX2 targets and splicing outcomes.

2021-06-23 | GSE140679 | GEO

RBFOX2 alter splicing outcome in distinct binding modes with multiple protein partners

Project description:RBFOX2 alter splicing outcome in distinct binding modes with multiple protein partners

| PRJNA590496 | ENA

Improved ligand-protein binding affinity predictions using multiple binding modes.

Project description:Accurate ligand-protein binding affinity prediction, for a set of similar binders, is a major challenge in the lead optimization stage in drug development. In general, docking and scoring functions perform unsatisfactorily in this application. Docking calculations, followed by molecular dynamics simulations and free energy calculations can be applied to improve the predictions. However, for targets with large, flexible binding sites, with no experimentally determined binding modes for a set of ligands, insufficient sampling can decrease the accuracy of the free energy calculations. Cytochrome P450s, a protein family of major importance for drug metabolism, is an example of a challenging target for binding affinity predictions. As a result, the choice of starting structure from the docking solutions becomes crucial. In this study, an iterative scheme is introduced that includes multiple independent molecular dynamics simulations to obtain weighted ensemble averages to be used in the linear interaction energy method. The proposed scheme makes the initial pose selection less crucial for further simulation, as it automatically calculates the relative weights of the various poses. It also properly takes into account the possibility that multiple binding modes contribute similarly to the overall affinity, or of similar compounds occupying very different poses. The method was applied to a set of 12 compounds binding to cytochrome P450 2C9 and it displayed a root mean-square error of 2.9 kJ/mol.

| S-EPMC2877349 | biostudies-literature

Rbfox2 controls autoregulation in RNA-binding protein networks.

Project description:The tight regulation of splicing networks is critical for organismal development. To maintain robust splicing patterns, many splicing factors autoregulate their expression through alternative splicing-coupled nonsense-mediated decay (AS-NMD). However, as negative autoregulation results in a self-limiting window of splicing factor expression, it is unknown how variations in steady-state protein levels can arise in different physiological contexts. Here, we demonstrate that Rbfox2 cross-regulates AS-NMD events within RNA-binding proteins to alter their expression. Using individual nucleotide-resolution cross-linking immunoprecipitation coupled to high-throughput sequencing (iCLIP) and mRNA sequencing, we identified >200 AS-NMD splicing events that are bound by Rbfox2 in mouse embryonic stem cells. These "silent" events are characterized by minimal apparent splicing changes but appreciable changes in gene expression upon Rbfox2 knockdown due to degradation of the NMD-inducing isoform. Nearly 70 of these AS-NMD events fall within genes encoding RNA-binding proteins, many of which are autoregulated. As with the coding splicing events that we found to be regulated by Rbfox2, silent splicing events are evolutionarily conserved and frequently contain the Rbfox2 consensus UGCAUG. Our findings uncover an unexpectedly broad and multilayer regulatory network controlled by Rbfox2 and offer an explanation for how autoregulatory splicing networks are tuned.

| S-EPMC3967051 | biostudies-literature

Optical tweezers experiments resolve distinct modes of DNA-protein binding.

Project description:Optical tweezers are ideally suited to perform force microscopy experiments that isolate a single biomolecule, which then provides multiple binding sites for ligands. The captured complex may be subjected to a spectrum of forces, inhibiting or facilitating ligand activity. In the following experiments, we utilize optical tweezers to characterize and quantify DNA binding of various ligands. High mobility group type B (HMGB) proteins, which bind to double-stranded DNA, are shown to serve the dual purpose of stabilizing and enhancing the flexibility of double stranded DNA. Unusual intercalating ligands are observed to thread into and lengthen the double-stranded structure. Proteins binding to both double- and single-stranded DNA, such as the alpha polymerase subunit of E. coli Pol III, are characterized, and the subdomains containing the distinct sites responsible for binding are isolated. Finally, DNA binding of bacteriophage T4 and T7 single-stranded DNA (ssDNA) binding proteins is measured for a range of salt concentrations, illustrating a binding model for proteins that slide along double-stranded DNA, ultimately binding tightly to ssDNA. These recently developed methods quantify both the binding activity of the ligand as well as the mode of binding.

| S-EPMC2753391 | biostudies-literature

Rbfox2 controls autoregulation in RNA binding protein networks

Project description:We generated a global analysis of Rbfox2 splicing regulation combined with a highly specific, single nucleotide-resolution Rbfox2 RNA binding map. We found that Rbfox2 regulates the splicing and expression of many previously unknown targets, and particularly a number of RNA binding proteins (RBPs), by modulating alternative splicing coupled-NMD. Based on our observations of RBP-Rbfox2 co-regulation with a polarity predicted by Rbfox2 binding, we propose a model whereby Rbfox2 tunes autoregulatory splicing events to control RBP expression levels and in turn alter their respective splicing networks. iCLIP for epitope-tagged Rbfox2 and control untagged Rbfox2; RNAseq of control and Rbfox2 knockdown in mouse embryonic stem cells

2014-03-17 | E-GEOD-54794 | biostudies-arrayexpress

Specificity of broad protein interaction surfaces for proteins with multiple binding partners.

Project description:Analysis of protein-protein interaction networks has revealed the presence of proteins with multiple interaction ligand proteins, such as hub proteins. For such proteins, multiple ligands would be predicted as interacting partners when predicting all-to-all protein-protein interactions (PPIs). In this work, to obtain a better understanding of PPI mechanisms, we focused on protein interaction surfaces, which differ between protein pairs. We then performed rigid-body docking to obtain information of interfaces of a set of decoy structures, which include many possible interaction surfaces between a certain protein pair. Then, we investigated the specificity of sets of decoy interactions between true binding partners in each case of alpha-chymotrypsin, actin, and cyclin-dependent kinase 2 as test proteins having multiple true binding partners. To observe differences in interaction surfaces of docking decoys, we introduced broad interaction profiles (BIPs), generated by assembling interaction profiles of decoys for each protein pair. After cluster analysis, the specificity of BIPs of true binding partners was observed for each receptor. We used two types of BIPs: those involved in amino acid sequences (BIP-seqs) and those involved in the compositions of interacting amino acid residue pairs (BIP-AAs). The specificity of a BIP was defined as the number of group members including all true binding partners. We found that BIP-AA cases were more specific than BIP-seq cases. These results indicated that the composition of interacting amino acid residue pairs was sufficient for determining the properties of protein interaction surfaces.

| S-EPMC5042157 | biostudies-literature

Functional characterization of three leishmania poly(a) binding protein homologues with distinct binding properties to RNA and protein partners.

Project description:Trypanosomatid protozoans are reliant on posttranscriptional processes to control gene expression. Regulation occurs at the levels of mRNA processing, stability, and translation, events that may require the participation of the poly(A) binding protein (PABP). Here, we have undertaken a functional study of the three distinct Leishmania major PABP (LmPABP) homologues: the previously described LmPABP1; LmPABP2, orthologous to the PABP described from Trypanosoma species; and LmPABP3, unique to Leishmania. Sequence identity between the three PABPs is no greater than 40%. In assays measuring binding to A-rich sequences, LmPABP1 binding was poly(A) sensitive but heparin insensitive; LmPABP2 binding was heparin sensitive and less sensitive to poly(A), compatible with unique substitutions observed in residues implicated in poly(A) binding; and LmPABP3 displayed intermediate properties. All three homologues are simultaneously expressed as abundant cytoplasmic proteins in L. major promastigotes, but only LmPABP1 is present as multiple isoforms. Upon transcription inhibition, LmPABP2 and -3 migrated to the nucleus, while LmPABP1 remained predominantly cytoplasmic. Immunoprecipitation assays showed an association between LmPABP2 and -3. Although the three proteins bound to a Leishmania homologue of the translation initiation factor eukaryotic initiation factor 4G (eIF4G) (LmEIF4G3) in vitro, LmPABP1 was the only one to copurify with native LmEIF4G3 from cytoplasmic extracts. Functionality was tested using RNA interference (RNAi) in Trypanosoma brucei, where both orthologues to LmPABP1 and -2 are required for cellular viability. Our results indicate that these homologues have evolved divergent functions, some of which may be unique to the trypanosomatids, and reinforces a role for LmPABP1 in translation through its interaction with the eIF4G homologue.

| S-EPMC2950419 | biostudies-literature

Two distinct binding modes of a protein cofactor with its target RNA.

Project description:Like most cellular RNA enzymes, the bI5 group I intron requires binding by a protein cofactor to fold correctly. Here, we use single-molecule approaches to monitor the structural dynamics of the bI5 RNA in real time as it assembles with its CBP2 protein cofactor. These experiments show that CBP2 binds to the target RNA in two distinct modes with apparently opposite effects: a "non-specific" mode that forms rapidly and induces large conformational fluctuations in the RNA, and a "specific" mode that forms slowly and stabilizes the native RNA structure. The bI5 RNA folds though multiple pathways toward the native state, typically traversing dynamic intermediate states induced by non-specific binding of CBP2. These results suggest that the protein cofactor-assisted RNA folding involves sequential non-specific and specific protein-RNA interactions. The non-specific interaction potentially increases the local concentration of CBP2 and the number of conformational states accessible to the RNA, which may promote the formation of specific RNA-protein interactions.

| S-EPMC2633024 | biostudies-other

Two distinct DNA binding modes guide dual roles of a CRISPR-Cas protein complex.

Project description:Small RNA-guided protein complexes play an essential role in CRISPR-mediated immunity in prokaryotes. While these complexes initiate interference by flagging cognate invader DNA for destruction, recent evidence has implicated their involvement in new CRISPR memory formation, called priming, against mutated invader sequences. The mechanism by which the target recognition complex mediates these disparate responses-interference and priming-remains poorly understood. Using single-molecule FRET, we visualize how bona fide and mutated targets are differentially probed by E. coli Cascade. We observe that the recognition of bona fide targets is an ordered process that is tightly controlled for high fidelity. Mutated targets are recognized with low fidelity, which is featured by short-lived and PAM- and seed-independent binding by any segment of the crRNA. These dual roles of Cascade in immunity with distinct fidelities underpin CRISPR-Cas robustness, allowing for efficient degradation of bona fide targets and priming of mutated DNA targets.

| S-EPMC4475636 | biostudies-literature

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