1. High-sensitivity in situ capture of endogenous RNA-protein interactions in fixed cells and primary tissues
- Author
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Liang, Qishan, Yu, Tao, Kofman, Eric, Jagannatha, Pratibha, Rhine, Kevin, Yee, Brian A, Corbett, Kevin D, and Yeo, Gene W
- Subjects
Biochemistry and Cell Biology ,Bioinformatics and Computational Biology ,Biological Sciences ,Genetics ,2.1 Biological and endogenous factors ,Generic health relevance ,Brain ,Animals ,Humans ,Mice ,RNA-Binding Proteins ,DNA-Binding Proteins ,Repressor Proteins ,RNA ,Binding Sites ,Protein Binding ,HEK293 Cells ,RNA Splicing Factors - 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.
- Published
- 2024