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

Qishan Liang, Tao Yu, Eric Kofman, Pratibha Jagannatha, Kevin Rhine, Brian A. Yee, Kevin D. Corbett () and Gene W. Yeo ()
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Qishan Liang: University of California San Diego
Tao Yu: University of California San Diego
Eric Kofman: University of California San Diego
Pratibha Jagannatha: University of California San Diego
Kevin Rhine: University of California San Diego
Brian A. Yee: University of California San Diego
Kevin D. Corbett: University of California San Diego
Gene W. Yeo: University of California San Diego

Nature Communications, 2024, vol. 15, issue 1, 1-16

Abstract: 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.

Date: 2024
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DOI: 10.1038/s41467-024-50363-4

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