scholarly journals Effective identification of RNA-binding proteins using RNA Immunoprecipitation v1 (protocols.io.bjpbkmin)

protocols.io ◽  
2020 ◽  
Author(s):  
Georgios I ◽  
Philip N
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Immunoprecipitation

scholarly journals RNA Binding Proteins Control Transdifferentiation of Hepatic Stellate Cells into Myofibroblasts

2018 ◽  
Vol 48 (3) ◽  
pp. 1215-1229 ◽  
Author(s):  
Sihyung Wang ◽  
Youngmi Jung ◽  
Jeongeun Hyun ◽  
Matthew Friedersdorf ◽  
Seh-Hoon Oh ◽  
...  
Keyword(s):  
Hepatic Stellate Cells ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Stellate Cells ◽  
Fibrous Matrix ◽  
Rna Immunoprecipitation ◽  
Physical Interactions ◽  
Β Receptor ◽  
Dependent Mechanism

Background/Aims: Myofibroblasts (MF) derived from quiescent nonfibrogenic hepatic stellate cells (HSC) are the major sources of fibrous matrix in cirrhosis. Because many factors interact to regulate expansion and regression of MF-HSC populations, efforts to prevent cirrhosis by targeting any one factor have had limited success, motivating research to identify mechanisms that integrate these diverse inputs. As key components of RNA regulons, RNA binding proteins (RBPs) may fulfill this function by orchestrating changes in the expression of multiple genes that must be coordinately regulated to affect the complex phenotypic modifications required for HSC transdifferentiation. Methods: We profiled the transcriptomes of quiescent and MF-HSC to identify RBPs that were differentially-expressed during HSC transdifferentiation, manipulated the expression of the most significantly induced RBP, insulin like growth factor 2 binding protein 3 (Igf2bp3), and evaluated transcriptomic and phenotypic effects. Results: Depleting Igf2bp3 changed the expression of thousands of HSC genes, including multiple targets of TGF-β signaling, and caused HSCs to reacquire a less proliferative, less myofibroblastic phenotype. RNA immunoprecipitation assays demonstrated that some of these effects were mediated by direct physical interactions between Igf2bp3 and mRNAs that control proliferative activity and mesenchymal traits. Inhibiting TGF-β receptor-1 signaling revealed a microRNA-dependent mechanism that induces Igf2bp3. Conclusions: The aggregate results indicate that HSC transdifferentiation is ultimately dictated by Igf2bp3-dependent RNA regulons and thus, can be controlled simply by manipulating Igf2bp3.

scholarly journals Interaction of Sox2 with RNA binding proteins in mouse embryonic stem cells

2019 ◽  
Author(s):  
Samudyata ◽  
Paulo P. Amaral ◽  
Pär G. Engström ◽  
Samuel C. Robson ◽  
Michael L. Nielsen ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Embryonic Stem ◽  
Messenger Rnas ◽  
Heterochromatin Protein 1 ◽  
Non Coding Rna ◽  
Rna Immunoprecipitation ◽  
Mes Cells

AbstractSox2 is a master transcriptional regulator of embryonic development. In this study, we determined the protein interactome of Sox2 in the chromatin and nucleoplasm of mouse embryonic stem (mES) cells. Apart from canonical interactions with pluripotency-regulating transcription factors, we identified interactions with several chromatin modulators, including members of the heterochromatin protein 1 (HP1) family, suggesting a role of Sox2 in chromatin-mediated transcriptional repression. Sox2 was also found to interact with RNA binding proteins (RBPs), including proteins involved in RNA processing. RNA immunoprecipitation followed by sequencing revealed that Sox2 associates with different messenger RNAs, as well as small nucleolar RNA Snord34 and the non-coding RNA 7SK. 7SK has been shown to regulate transcription at regulatory regions, which could suggest a functional interaction with Sox2 for chromatin recruitment. Nevertheless, we found no evidence of Sox2 modulating recruitment of 7SK to chromatin when examining 7SK chromatin occupancy by Chromatin Isolation by RNA Purification (ChIRP) in Sox2 depleted mES cells. In addition, knockdown of 7SK in mES cells did not lead to any change in Sox2 occupancy at 7SK-regulated genes. Thus, our results show that Sox2 extensively interact with RBPs, and suggest that Sox2 and 7SK co-exist in a ribonucleoprotein complex whose function is not to regulate chromatin recruitment, but might rather regulate other processes in the nucleoplasm.Summary blurbSox2 interacts with RNA-binding proteins and diverse RNAs

scholarly journals Validation and classification of RNA binding proteins identified by mRNA interactome capture

2021 ◽  
Author(s):  
Vaishali ◽  
Lyudmila Dimitrova-Paternoga ◽  
Kevin Haubrich ◽  
Mai Sun ◽  
Anne Ephrussi ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Modular Organization ◽  
Binding Domains ◽  
Mammalian Cell Lines ◽  
Rna Immunoprecipitation ◽  
Rna Binding Domains ◽  
Rna Binders

AbstractRNA binding proteins (RBPs) take part in all steps of the RNA life cycle and are often essential for cell viability. Most RBPs have a modular organization and comprise a set of canonical RNA binding domains. However, in recent years a number of high-throughput mRNA interactome studies on yeast, mammalian cell lines and whole organisms have uncovered a multitude of novel mRNA interacting proteins that lack classical RNA binding domains. Whereas a few have been confirmed to be direct and functionally relevant RNA binders, biochemical and functional validation of RNA binding of most others is lacking. In this study, we employed a combination of NMR spectroscopy and biochemical studies to test the RNA binding properties of six putative RNA binding proteins. Half of the analysed proteins showed no interaction, whereas the other half displayed weak chemical shift perturbations upon titration with RNA. One of the candidates we found to interact weakly with RNA in vitro is Drosophila melanogaster End binding protein 1 (EB1), a master regulator of microtubule plus-end dynamics. Further analysis showed that EB1’s RNA binding occurs on the same surface as that with which EB1 interacts with microtubules. RNA immunoprecipitation and colocalization experiments suggest that EB1 is a rather non-specific, opportunistic RNA binder. Our data suggest that care should be taken when embarking on an RNA binding study involving these unconventional, novel RBPs, and we recommend initial and simple in vitro RNA binding experiments.

scholarly journals Discovering the Interactions between Circular RNAs and RNA-binding Proteins from CLIP-seq Data using circScan

2017 ◽  
Author(s):  
Bin Li ◽  
Xiao-Qin Zhang ◽  
Shu-Rong Liu ◽  
Shun Liu ◽  
Wen-Ju Sun ◽  
...  
Keyword(s):  
Binding Proteins ◽  
High Throughput Sequencing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Initiation Factor ◽  
Circular Rnas ◽  
New Approach ◽  
Rna Immunoprecipitation ◽  
Mammalian Genomes ◽  
Novel Interactions

AbstractAlthough tens of thousands of circular RNAs (circRNAs) have been identified in mammalian genomes, only few of them have been characterized with biological functions. Here, we report a new approach, circScan, to identify regulatory interactions between circRNAs and RNA-binding proteins (RBPs) by discovering back-splicing reads from Cross-Linking and Immunoprecipitation followed by high-throughput sequencing (CLIP-seq) data. By using our method, we have systematically scanned ~1500 CLIP-seq datasets, and identified ~12540 and ~1090 novel circRNA-RBP interactions in human and mouse genomes, respectively, which include all known interactions between circRNAs and Argonaute (AGO) proteins. More than twenty novel interactions were further experimentally confirmed by RNA Immunoprecipitation quantitative PCR (RIP-qPCR). Importantly, we uncovered that some natural circRNAs interacted with cap-independent translation factors eukaryotic initiation factor 3 (eIF3) and N6-Methyladenosine (m6A), indicating they can be translated into proteins. These findings demonstrate that circRNAs are regulated by various RBPs, suggesting they may play important roles in diverse biological processes.

scholarly journals Cohesin SA1 and SA2 are RNA binding proteins that localize to RNA containing regions on DNA

2020 ◽  
Vol 48 (10) ◽  
pp. 5639-5655 ◽  
Author(s):  
Hai Pan ◽  
Miao Jin ◽  
Ashwin Ghadiyaram ◽  
Parminder Kaur ◽  
Henry E Miller ◽  
...  
Keyword(s):  
Single Molecule ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Strand Break ◽  
Data Sets ◽  
Cohesin Complex ◽  
Replication Fork Progression ◽  
Chromatin Immunoprecipitation Sequencing ◽  
Rna Immunoprecipitation

Abstract Cohesin SA1 (STAG1) and SA2 (STAG2) are key components of the cohesin complex. Previous studies have highlighted the unique contributions by SA1 and SA2 to 3D chromatin organization, DNA replication fork progression, and DNA double-strand break (DSB) repair. Recently, we discovered that cohesin SA1 and SA2 are DNA binding proteins. Given the recently discovered link between SA2 and RNA-mediated biological pathways, we investigated whether or not SA1 and SA2 directly bind to RNA using a combination of bulk biochemical assays and single-molecule techniques, including atomic force microscopy (AFM) and the DNA tightrope assay. We discovered that both SA1 and SA2 bind to various RNA containing substrates, including ssRNA, dsRNA, RNA:DNA hybrids, and R-loops. Importantly, both SA1 and SA2 localize to regions on dsDNA that contain RNA. We directly compared the SA1/SA2 binding and R-loops sites extracted from Chromatin Immunoprecipitation sequencing (ChIP-seq) and DNA-RNA Immunoprecipitation sequencing (DRIP-Seq) data sets, respectively. This analysis revealed that SA1 and SA2 binding sites overlap significantly with R-loops. The majority of R-loop-localized SA1 and SA2 are also sites where other subunits of the cohesin complex bind. These results provide a new direction for future investigation of the diverse biological functions of SA1 and SA2.

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