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Footprinting SHAPE-eCLIP reveals transcriptome-wide hydrogen bonds at RNA-protein interfaces

ABSTRACT: Footprinting SHAPE-eCLIP reveals transcriptome-wide hydrogen bonds at RNA-protein interfaces

PROVIDER: PRJNA630074 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11682352.fastq.gz	Fastqsanger.gz
	SRR11682353.fastq.gz	Fastqsanger.gz
	SRR11682354.fastq.gz	Fastqsanger.gz
	SRR11682355.fastq.gz	Fastqsanger.gz
	SRR11682356.fastq.gz	Fastqsanger.gz

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Footprinting SHAPE-eCLIP reveals transcriptome-wide hydrogen bonds at RNA-protein interfaces

Project description:Discovering the interaction mechanism and location of RNA binding proteins (RBPs) on RNA is critical for understanding gene expression regulation. Here, we apply selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) on in vivo transcripts to identify transcriptome-wide footprints (fSHAPE) on RNA when compared to protein-absent transcripts. Structural analyses indicate that fSHAPE precisely detects nucleotides whose base moieties hydrogen bond with protein and that fSHAPE patterns can predict binding sites of RBPs of interest. To illustrate, we demonstrate that fSHAPE correctly identifies known and novel iron response protein RNA elements. Furthermore, we enable selective interrogation of RNA-protein complexes by integrating SHAPE and fSHAPE with crosslinking and immunoprecipitation (eCLIP) of desired RBPs. To demonstrate, histone stem loop elements and their nucleotides that hydrogen bond with stem-loop binding protein were identified by SHAPE-eCLIP and fSHAPE-eCLIP. Together, these technologies greatly expand on strategies for understanding specific cellular RNA interactions in RNA-protein complexes.

2020-11-30 | GSE149767 | GEO

Footprinting SHAPE-eCLIP Reveals Transcriptome-wide Hydrogen Bonds at RNA-Protein Interfaces.

Project description:Discovering the interaction mechanism and location of RNA-binding proteins (RBPs) on RNA is critical for understanding gene expression regulation. Here, we apply selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) on in vivo transcripts compared to protein-absent transcripts in four human cell lines to identify transcriptome-wide footprints (fSHAPE) on RNA. Structural analyses indicate that fSHAPE precisely detects nucleobases that hydrogen bond with protein. We demonstrate that fSHAPE patterns predict binding sites of known RBPs, such as iron response elements in both known loci and previously unknown loci in CDC34, SLC2A4RG, COASY, and H19. Furthermore, by integrating SHAPE and fSHAPE with crosslinking and immunoprecipitation (eCLIP) of desired RBPs, we interrogate specific RNA-protein complexes, such as histone stem-loop elements and their nucleotides that hydrogen bond with stem-loop-binding proteins. Together, these technologies greatly expand our ability to study and understand specific cellular RNA interactions in RNA-protein complexes.

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Carbene footprinting reveals binding interfaces of a multimeric membrane spanning protein

Project description:Mapping the interaction sites between membrane spanning proteins is a key challenge in structural biology. Here we develop and apply carbene footprinting technology to identify the interfacial sites of a trimeric membrane protein. We show how the footprinting probe is effectively incorporated into detergent micelles leading to efficient labelling of the external membrane-spanning regions of the protein.

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fSHAPE, fSHAPE-eCLIP, and SHAPE-eCLIP probe transcript regions that interact with specific proteins.

Project description:Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) structure probing techniques characterize the secondary structure of RNA molecules, which influence their functions and interactions. A variation of SHAPE, footprinting SHAPE (fSHAPE), probes RNA in the presence and absence of protein to identify RNA bases that hydrogen-bond with protein. SHAPE or fSHAPE coupled with enhanced crosslinking and immunoprecipitation (SHAPE-eCLIP or fSHAPE-eCLIP) pulls down RNAs bound by any protein of interest and returns their structure or protein interaction information, respectively. Here, we describe detailed protocols for SHAPE-eCLIP and fSHAPE-eCLIP and an analysis protocol for fSHAPE. For complete details on the use and execution of these protocols, please refer to Corley et al. (2020).

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