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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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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: Not available

| S-EPMC8074864 | biostudies-literature

Carbene Footprinting Accurately Maps Binding Sites in Protein-Ligand and Protein-Protein Interactions

Project description:Specific interactions between proteins and their binding partners are fundamental to life processes. Differential protein footprinting using a new and efficient, photo-activated aryltrifluromethylcarbene probe together with mass spectrometry has been employed to identify protein-ligand and protein-protein interaction sites. In a model protein-small molecule system, the location of a penta-N-acetylchitopentaose carbohydrate substrate was accurately mapped to the binding cleft of lysozyme. As a fine detail, footprinting revealed disruption of an intramolecular hydrogen-bond network within lysozyme, which is known to be associated with substrate binding. In a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5, and a diubiquitin substrate were located to different functional domains thus clarifying uncertainties from an X-ray crystal structure of USP5. The much improved properties of this bespoke diazirine probe make differential carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.

2016-12-19 | PXD004971 | Pride

RBM33 eCLIP

Project description:RBM33 eCLIP

| PRJNA791310 | ENA

Enhanced CLIP uncovers IMP protein-RNA targets in human pluripotent stem cells important for cell adhesion and survival [eCLIP-Seq]

Project description:Human pluripotent stem cells (hPSCs) require precise control of post-transcriptional RNA networks to maintain proliferation and survival. Using a recently developed enhanced UV crosslinking and immunoprecipitation (eCLIP) approach, we identify RNA targets of the IMP/IGF2BP family of RNA-binding proteins in hPSCs. At the broad region- and binding site-level IMP1 and IMP2 show reproducible binding to a large and overlapping set of 3'UTR-enriched targets. RNA Bind-N-Seq applied to recombinant full-length IMP1 and IMP2 reveals CA-rich motifs that are enriched in eCLIP-defined binding sites. We observe that IMP1 loss in hPSCs recapitulates IMP1 phenotypes, including a reduction in cell adhesion and an increase in cell death. For cell adhesion, in hPSCs we find IMP1 maintains levels of integrin mRNA, specifically regulating RNA stability of ITGB5. Additionally, we show IMP1 can be linked to hPSC survival via direct target BCL2. Thus, transcriptome-wide binding profiles identify hPSC targets modulating well-characterized IMP1 roles. eCLIP-seq was performed in biological replicate for IGF2BP1/IMP1 and IGF2BP2/IMP2, as well as one replicate each for IGF2BP3/IMP3, RBFOX2, and an IgG control. Each sample has a size-matched input control for analysis

2016-04-07 | E-GEOD-78507 | biostudies-arrayexpress

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

Project description: Not available

| S-EPMC8406031 | biostudies-literature

Nanoparticles and photochemistry for Native-like transmembrane protein footprinting

Project description:Mass spectrometry based footprinting can probe higher order structure of proteins. We bond photocatalytic nanoparticles to a lipid bilayer that, upon laser irradiation, produce high local concentrations of radicals that penetrate the lipid layer made permeable by a simultaneous laser-initiated PB reaction. This approach achieves high footprinting coverage for membrane proteins in liposomes, helps locate both ligand-binding residues in a transporter and ligand-induced conformational changes, and reveals structural aspects of the flexible unbound state. Overall, this approach proves highly effective in intramembrane footprinting and forges a connection between materials science and biology.

2021-09-26 | PXD027819 | Pride

ENO1's RNA-binding sites in Human HeLa cells using eCLIP

Project description:eCLIP was performed for ENO1 in HeLa cells, following the protocol described by Van Nostrand et al. (2016). Libraries for six immunoprecipitation and size-matched input controls were produced. In addition, libraries were produced for two no-crosslinking controls. The libraries were sequenced using paired-end sequencing (PE125) on an Illumina HiSeq2000 platform.

2020-10-27 | E-MTAB-9031 | biostudies-arrayexpress

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.

2017-09-29 | PXD007207 | Pride

Ribosome footprinting of fly heads

Project description:We applied ribosomal footprinting experiment followed by RNA-seq in order to detect circular RNA associated with ribosomes in fly heads.

2017-03-24 | GSE79626 | GEO

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