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High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues.

ABSTRACT: RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. The robust, versatile, and sensitive INSCRIBE workflow is particularly beneficial for precious tissues such as clinical samples, empowering the exploration of genuine RBP-RNA interactions in RNA-related disease contexts.

SUBMITTER: Liang Q

PROVIDER: S-EPMC11329496 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues.

Liang Qishan Q Yu Tao T Kofman Eric E Jagannatha Pratibha P Rhine Kevin K Yee Brian A BA Corbett Kevin D KD Yeo Gene W GW

Nature communications 20240816 1

RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across ...[more]

PMID: 39152130

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Dataset Information

High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues.

Publications

High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues.

Similar Datasets

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