Dataset Information

Improved Analysis of RNA Localization by Spatially Restricted Oxidation of RNA-Protein Complexes.

ABSTRACT: Recent analysis of transcriptomes has revealed that RNAs perform a myriad of functions beyond encoding proteins. Critical to RNA function is its transport to unique subcellular locations. Despite the importance of RNA localization, it is still very challenging to study in an unbiased manner. We recently described the ability to tag RNA molecules within subcellular locations through spatially restricted nucleobase oxidation. Herein, we describe a dramatic improvement of this protocol through the localized oxidation and tagging of proteins. Isolation of RNA-protein complexes enabled the enrichment of challenging RNA targets on chromatin and presented a considerably optimized protocol for the analysis of RNA subcellular localization within living cells.

SUBMITTER: Li Y

PROVIDER: S-EPMC6234203 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Publications

Improved Analysis of RNA Localization by Spatially Restricted Oxidation of RNA-Protein Complexes.

Li Ying Y Aggarwal Mahima B MB Ke Ke K Nguyen Kim K Spitale Robert C RC

Biochemistry 20180227 10

Recent analysis of transcriptomes has revealed that RNAs perform a myriad of functions beyond encoding proteins. Critical to RNA function is its transport to unique subcellular locations. Despite the importance of RNA localization, it is still very challenging to study in an unbiased manner. We recently described the ability to tag RNA molecules within subcellular locations through spatially restricted nucleobase oxidation. Herein, we describe a dramatic improvement of this protocol through the ...[more]

PMID: 29474061

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Project description:Protein complexes are not static, but rather highly dynamic with subunits that undergo 1-dimensional diffusion with respect to each other. Interactions within protein complexes are modulated through regulatory inputs that alter interactions and introduce new components and deplete existing components through exchange. While it is clear that the structure and function of any given protein complex is coupled to its dynamical properties, it remains a challenge to predict the possible conformations that complexes can adopt. Protein-fragment Complementation Assays detect physical interactions between protein pairs constrained to ≤8 nm from each other in living cells. This method has been used to build networks composed of 1000s of pair-wise interactions. Significantly, these networks contain a wealth of dynamic information, as the assay is fully reversible and the proteins are expressed in their natural context. In this study, we describe a method that extracts this valuable information in the form of predicted conformations, allowing the user to explore the conformational landscape, to search for structures that correlate with an activity state, and estimate the abundance of conformations in the living cell. The generator is based on a Markov Chain Monte Carlo simulation that uses the interaction dataset as input and is constrained by the physical resolution of the assay. We applied this method to an 18-member protein complex composed of the seven core proteins of the budding yeast Arp2/3 complex and 11 associated regulators and effector proteins. We generated 20,480 output structures and identified conformational states using principle component analysis. We interrogated the conformation landscape and found evidence of symmetry breaking, a mixture of likely active and inactive conformational states and dynamic exchange of the core protein Arc15 between core and regulatory components. Our method provides a novel tool for prediction and visualization of the hidden dynamics within protein interaction networks.

Dataset Information

Improved Analysis of RNA Localization by Spatially Restricted Oxidation of RNA-Protein Complexes.

Publications

Improved Analysis of RNA Localization by Spatially Restricted Oxidation of RNA-Protein Complexes.

Similar Datasets

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