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

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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RNA Binding Proteins Control Transdifferentiation of Hepatic Stellate Cells into Myofibroblasts

Cellular Physiology and Biochemistry ◽

10.1159/000491987 ◽

2018 ◽

Vol 48 (3) ◽

pp. 1215-1229 ◽

Cited By ~ 5

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.

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

10.1101/560383 ◽

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

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Predicting localized affinity of RNA binding proteins to transcripts with convolutional neural networks

10.1101/2021.06.02.446817 ◽

2021 ◽

Author(s):

Alexander Kitaygorodsky ◽

Emily Jin ◽

Yufeng Shen

Keyword(s):

Binding Affinity ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Specific Binding ◽

Computational Prediction ◽

Data Sets ◽

Coding Region ◽

Protein Coding ◽

Specific Affinity

RNA binding proteins (RBPs) are important regulators of transcriptional and post-transcriptional processes. Computational prediction of localized RBP binding affinity with transcripts is important for interpretation of genetic variation, especially variants outside of protein coding region. Here we describe POLARIS (Prediction Of Localized Affinity for RBPs In Sequence), a new deep-learning method for achieving fast, site-specific binding affinity predictions of RNA-binding proteins (RBPs) to the transcribed genome. POLARIS has two modules: 1. a convolutional neural network (CNN) to predict overall RBP binding within a region based on transcript sequence content and expression level; 2. a Gradient-weighted Class Activation Mapping (GradCAM) implementation for efficient signal backpropagation to individual sequence positions. We trained the model using enhanced crosslinking and immunoprecipitation (eCLIP) data from ENCODE. POLARIS has good performance with a median AUC ~ 0.96 for 160 RBPs across three different cell lines, substantially higher than selected popular published methods trained and tested on the same data sets. When tested on data from a different cell line with the same RBPs, the overall performance is maintained, supporting the ability of cell-type specific affinity prediction. Finally, the GradCAM module allows the model to identify the informative sites in a region that drive prediction. The localized prediction facilitates interpretation of the results and provides basis for inference of functional impact of noncoding variants.

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New Faces of old Friends: Emerging new Roles of RNA-Binding Proteins in the DNA Double-Strand Break Response

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.668821 ◽

2021 ◽

Vol 8 ◽

Author(s):

Julie A. Klaric ◽

Stas Wüst ◽

Stephanie Panier

Keyword(s):

Cellular Response ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Genome Instability ◽

Strand Break ◽

Dna Lesions ◽

Cell Cycle Checkpoints ◽

Dna Double Strand Breaks ◽

Dna Strand

DNA double-strand breaks (DSBs) are highly cytotoxic DNA lesions. To protect genomic stability and ensure cell homeostasis, cells mount a complex signaling-based response that not only coordinates the repair of the broken DNA strand but also activates cell cycle checkpoints and, if necessary, induces cell death. The last decade has seen a flurry of studies that have identified RNA-binding proteins (RBPs) as novel regulators of the DSB response. While many of these RBPs have well-characterized roles in gene expression, it is becoming increasingly clear that they also have non-canonical functions in the DSB response that go well beyond transcription, splicing and mRNA processing. Here, we review the current understanding of how RBPs are integrated into the cellular response to DSBs and describe how these proteins directly participate in signal transduction, amplification and repair at damaged chromatin. In addition, we discuss the implications of an RBP-mediated DSB response for genome instability and age-associated diseases such as cancer and neurodegeneration.

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Single-molecule imaging reveals dynamic biphasic partition of RNA-binding proteins in stress granules

The Journal of Cell Biology ◽

10.1083/jcb.201709007 ◽

2018 ◽

Vol 217 (4) ◽

pp. 1303-1318 ◽

Cited By ~ 44

Author(s):

Benedikt Niewidok ◽

Maxim Igaev ◽

Abel Pereira da Graca ◽

Andre Strassner ◽

Christine Lenzen ◽

...

Keyword(s):

Liquid Phase ◽

Single Molecule ◽

Mammalian Cells ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Liquid Droplet ◽

Binding Protein ◽

Stress Granules

Stress granules (SGs) are cytosolic, nonmembranous RNA–protein complexes. In vitro experiments suggested that they are formed by liquid–liquid phase separation; however, their properties in mammalian cells remain unclear. We analyzed the distribution and dynamics of two paradigmatic RNA-binding proteins (RBPs), Ras GTPase-activating protein SH3-domain–binding protein (G3BP1) and insulin-like growth factor II mRNA-binding protein 1 (IMP1), with single-molecule resolution in living neuronal cells. Both RBPs exhibited different exchange kinetics between SGs. Within SGs, single-molecule localization microscopy revealed distributed hotspots of immobilized G3BP1 and IMP1 that reflect the presence of relatively immobile nanometer-sized nanocores. We demonstrate alternating binding in nanocores and anomalous diffusion in the liquid phase with similar characteristics for both RBPs. Reduction of low-complexity regions in G3BP1 resulted in less detectable mobile molecules in the liquid phase without change in binding in nanocores. The data provide direct support for liquid droplet behavior of SGs in living cells and reveal transient binding of RBPs in nanocores. Our study uncovers a surprising disconnect between SG partitioning and internal diffusion and interactions of RBPs.

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Single-molecule imaging reveals translation of mRNAs localized to stress granules

10.1101/2020.03.31.018093 ◽

2020 ◽

Author(s):

Daniel Mateju ◽

Bastian Eichenberger ◽

Jan Eglinger ◽

Gregory Roth ◽

Jeffrey A. Chao

Keyword(s):

Single Molecule ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Cellular Stress ◽

Mrna Translation ◽

Stress Granules ◽

Living Cells ◽

Single Molecule Imaging ◽

Direct Role

SUMMARYCellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins. Although the function of SGs remains largely unknown, it is widely assumed they contain exclusively nontranslating mRNA. Here we re-examine this hypothesis using single-molecule imaging of mRNA translation in living cells. While our data confirms that non-translating mRNAs are preferentially recruited to SGs, we find unequivocal evidence for translation of mRNA localized to SGs. Our data indicate that SG-associated translation is not rare and that the entire translation cycle (initiation, elongation and termination) can occur for these transcripts. Furthermore, translating mRNAs can be observed transitioning between the cytosol and SGs without changing their translational status. Together, these results argue against a direct role for SGs in inhibition of mRNA translation.

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

10.1101/2021.02.02.429302 ◽

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.

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

10.1101/115980 ◽

2017 ◽

Cited By ~ 6

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.

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