Exonuclease assisted mapping of protein-RNA interactions (ePRINT)
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE230097 | GEO | 2024/03/04
REPOSITORIES: GEO
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Project description:RNA processing is a fundamental mode of gene regulation that is perturbed in a variety of diseases including cancer and neurodegenerative disorders. RNA-binding proteins (RBPs) regulate key aspects of RNA processing including alternative splicing, mRNA degradation and localization by physically binding RNA molecules. Current methods to map these interactions, such as CLIP, rely on purifying single proteins at a time. We have developed a new method (ePRINT) to map RBP-RNA interaction networks on a global scale without purifying individual RBPs. By deploying the exoribonuclease XRN1, ePRINT allows precise mapping of the 5’ end of the RBP binding site and uncovers direct and indirect targets of an RBP of interest. Importantly, ePRINT can also uncover RBPs that are differentially activated between cell fate transitions, including neural progenitor differentiation into neurons. Given its versatility, ePRINT has vast application potential as an investigative tool for RNA regulation in development, health and disease.
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Project description:RNAs are continuously associated with RNA-binding proteins (RBPs), and these interactions are necessary for many key cellular processes ranging from splicing to chromatin regulation. Although numerous approaches have been developed to map RNA-binding sites of individual RBPs, few methods exist that allow assessment of global RBP-RNA interactions. Here, we describe a universal, high-throughput, ribonuclease-mediated protein footprint sequencing approach that reveals RNA-protein interaction sites throughout a transcriptome of interest. We apply this method to the HeLa transcriptome and compare RBP binding sites found using different cross-linkers and ribonucleases. From this analysis, we identify numerous putative RBP binding motifs, reveal novel insights into co-binding by RBPs, and uncover a significant enrichment for disease-associated polymorphisms within RBP interaction sites.
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Project description:In our cells, a limited number of RNA binding proteins (RBPs) are responsible for all aspects of RNA metabolism across the entire transcriptome. To accomplish this, RBPs form regulatory units that act on specific target regulons. However, the landscape of RBP combinatorial interactions remains poorly explored. Here, we performed a systematic annotation of RBP combinatorial interactions via multimodal data integration. We built a large-scale map of RBP protein neighborhoods by generating in vivo proximity-dependent biotinylation datasets of 50 human RBPs. In parallel, we used CRISPR interference with single-cell readout to capture transcriptomic changes upon RBP knockdowns. By combining these physical and functional interaction readouts, along with the atlas of RBP mRNA targets from eCLIP assays, we generated an integrated map of functional RBP interactions. We then used this map to match RBPs to their context-specific functions and validated the predicted functions biochemically for four RBPs. This study highlights the previously underappreciated scale of the inter-RBP interactions, be it genetic or physical, and is a first step towards a more comprehensive understanding of post-transcriptional regulatory processes and their underlying molecular grammar.
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Project description:RNA-binding proteins (RBPs) are multifunctional regulators of gene expression with complex and context-dependent mechanisms of action, yet the regulatory grammar that underlies RBP-mediated functions remains underexplored. Here, we performed a multi-omic data integration to systematically decipher the context-specific functions of RBPs. First, we report a compendium of in vivo proximity-dependent biotinylation (BioID) datasets of 50 human RBPs, which we used to generate a large-scale map of RBP protein neighborhoods. In parallel, we took advantage of CRISPR-interference with single-cell RNA-seq read-out (Perturb-seq) to capture rich transcriptomic phenotypes downstream of each RBP knockdown. By combining these physical and functional interaction readouts, along with the ENCODE transcriptome-wide atlas of RBP binding sites from eCLIP assays, we generated an integrated map of functional RBP interactions. This map not only captures well-studied post-transcriptional processes, but also reveals numerous previously unknown RBP-mediated regulatory functions. For a number of these cases, we have validated their predicted context-specific RBP functions using biochemical and genetic approaches. By deciphering these complex modes of functional interactions between RBPs, we have taken the first step towards a more comprehensive understanding of post-transcriptional regulatory processes and their underlying molecular grammar.
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Project description:RNA-binding proteins (RBPs) are multifunctional regulators of gene expression with complex and context-dependent mechanisms of action, yet the regulatory grammar that underlies RBP-mediated functions remains underexplored. Here, we performed a multi-omic data integration to systematically decipher the context-specific functions of RBPs. First, we report a compendium of in vivo proximity-dependent biotinylation (BioID) datasets of 50 human RBPs, which we used to generate a large-scale map of RBP protein neighborhoods. In parallel, we took advantage of CRISPR-interference with single-cell RNA-seq read-out (Perturb-seq) to capture rich transcriptomic phenotypes downstream of each RBP knockdown. By combining these physical and functional interaction readouts, along with the ENCODE transcriptome-wide atlas of RBP binding sites from eCLIP assays, we generated an integrated map of functional RBP interactions. This map not only captures well-studied post-transcriptional processes, but also reveals numerous previously unknown RBP-mediated regulatory functions. For a number of these cases, we have validated their predicted context-specific RBP functions using biochemical and genetic approaches. By deciphering these complex modes of functional interactions between RBPs, we have taken the first step towards a more comprehensive understanding of post-transcriptional regulatory processes and their underlying molecular grammar.
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Project description:RNA-binding proteins (RBPs) are multifunctional regulators of gene expression with complex and context-dependent mechanisms of action, yet the regulatory grammar that underlies RBP-mediated functions remains underexplored. Here, we performed a multi-omic data integration to systematically decipher the context-specific functions of RBPs. First, we report a compendium of in vivo proximity-dependent biotinylation (BioID) datasets of 50 human RBPs, which we used to generate a large-scale map of RBP protein neighborhoods. In parallel, we took advantage of CRISPR-interference with single-cell RNA-seq read-out (Perturb-seq) to capture rich transcriptomic phenotypes downstream of each RBP knockdown. By combining these physical and functional interaction readouts, along with the ENCODE transcriptome-wide atlas of RBP binding sites from eCLIP assays, we generated an integrated map of functional RBP interactions. This map not only captures well-studied post-transcriptional processes, but also reveals numerous previously unknown RBP-mediated regulatory functions. For a number of these cases, we have validated their predicted context-specific RBP functions using biochemical and genetic approaches. By deciphering these complex modes of functional interactions between RBPs, we have taken the first step towards a more comprehensive understanding of post-transcriptional regulatory processes and their underlying molecular grammar.
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Project description:Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP “target” RNAs. To address this, an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP, was developed. easyCLIP, provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize unamplified libraries. 213 cross-link measurements were performed and found that RBPs could be distinguished from non-RBPs by RNA cross-link rates and that target RNAs could be defined as those with a complex frequency unlikely for a random protein. easyCLIP was used to study the 33 most recurrent cancer mutations across 28 RBPs. This demonstrated increased RNA binding per RBP molecule for KHDRBS2 and A1CF cancer mutants, and showed that the PCBP1L100P/Q cancer mutation also dramatically increased RNA binding. Quantitating RBP-RNA interactions can thus categorize proteins as RBPs or not and define the impact of specific disease-associated RBP mutations on RNA binding.
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