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High-density functional-RNA arrays as a versatile platform for studying RNA-based interactions.

ABSTRACT: We are just beginning to unravel the myriad of interactions in which non-coding RNAs participate. The intricate RNA interactome is the foundation of many biological processes, including bacterial virulence and human disease, and represents unexploited resources for the development of potential therapeutic interventions. However, identifying specific associations of a given RNA from the multitude of possible binding partners within the cell requires robust high-throughput systems for their rapid screening. Here, we present the first demonstration of functional-RNA arrays as a novel platform technology designed for the study of such interactions using immobilized, active RNAs. We have generated high-density RNA arrays by an innovative method involving surface-capture of in vitro transcribed RNAs. This approach has significant advantages over existing technologies, particularly in its versatility in regards to binding partner character. Indeed, proof-of-principle application of RNA arrays to both RNA-small molecule and RNA-RNA pairings is demonstrated, highlighting their potential as a platform technology for mapping RNA-based networks and for pharmaceutical screening. Furthermore, the simplicity of the method supports greater user-accessibility over currently available technologies. We anticipate that functional-RNA arrays will find broad utility in the expanding field of RNA characterization.

SUBMITTER: Phillips JO

PROVIDER: S-EPMC6101487 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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High-density functional-RNA arrays as a versatile platform for studying RNA-based interactions.

Phillips Jack O JO Butt Louise E LE Henderson Charlotte A CA Devonshire Martin M Healy Jess J Conway Stuart J SJ Locker Nicolas N Pickford Andrew R AR Vincent Helen A HA Callaghan Anastasia J AJ

Nucleic acids research 20180801 14

We are just beginning to unravel the myriad of interactions in which non-coding RNAs participate. The intricate RNA interactome is the foundation of many biological processes, including bacterial virulence and human disease, and represents unexploited resources for the development of potential therapeutic interventions. However, identifying specific associations of a given RNA from the multitude of possible binding partners within the cell requires robust high-throughput systems for their rapid ...[more]

PMID: 29846708

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Project description:High-density lipoprotein (HDL) is a mixture of complex particles mediating reverse cholesterol transport (RCT) and several cytoprotective activities. Despite its relevance for human health, many aspects of HDL-mediated lipid trafficking and cellular signaling remain elusive at the mo-lecular level. During HDL’s journey throughout the body, its functions are mediated through in-teractions with cell surface receptors on different cell types. To characterize and better under-stand the functional interplay between HDL particles and tissue, we analyzed the sur-faceome-residing receptor neighborhoods with which HDL potentially interacts. We applied a combination of chemoproteomic technologies including automated cell surface capturing (au-to-CSC) and HATRIC-based ligand–receptor capturing (HATRIC-LRC) on four different cellular model systems mimicking tissues relevant for RCT. The surfaceome analysis of EA.hy926, HEPG2, foam cells, and human aortic endothelial cells (HAECs) revealed the main currently known HDL scavenger receptor B1 (SCRB1), as well as 155 shared cell surface receptors repre-senting potential HDL-receptor interaction candidates . Since vascular endotheli-al growth factor A (VEGF-A) was recently found as a regulatory factor of transendothelial transport of HDL, we next analyzed the VEGF-modulated surfaceome of HAEC using the au-to-CSC technology. VEGF-A treatment led to the remodeling of the surfaceome of HAEC cells, including the previously reported higher surfaceome abundance of SCRB1. In total, 165 addition-al receptors were found on HAEC upon VEGF-A treatment representing SCRB1 co-regulated re-ceptors potentially involved in HDL function. Using the HATRIC-LRC technology on human endothelial cells, we specifically aimed for the identification of other bona fide (co-)receptors of HDL beyond SCRB1. HATRIC-LRC enabled, next to SCRB1, the identification of the receptor ty-rosine-protein kinase Mer (MERTK). Through RNA interference, we revealed its contribution to endothelial HDL binding and uptake. Furthermore, subsequent proximity ligation assays (PLAs) demonstrated the spatial vicinity of MERTK and SCRB1 on the endothelial cell surface. The data shown provide direct evidence for a complex and dynamic HDL receptome and that receptor nanoscale organization may influence binding and uptake of HDL.

Dataset Information

High-density functional-RNA arrays as a versatile platform for studying RNA-based interactions.

Publications

High-density functional-RNA arrays as a versatile platform for studying RNA-based interactions.

Similar Datasets

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