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eSymposia | Proteomics in Cell Biology and Disease


Dissect RNA-Protein Interactome in Living Cells Using CRISPR-Assisted Proximity Labeling


Sep 21, 2020 12:00am ‐ Sep 21, 2020 12:00am

Description

Dissect RNA-Protein Interactome in Living Cells Using CRISPR-Assisted Proximity Labeling Delineating the protein network associated with long non-coding RNAs (lncRNAs) is fundamental to understanding the functional mechanisms of lncRNAs. Current methods to identify lncRNA binding proteins either rely on crosslinking mediated complex co-precipitation or require extensive molecular engineering, leading to drawbacks such as loss of cellular context and low capture efficiency. We developed CRISPR-Assisted RNA-Protein Interaction Detection (CARPID), which leverages CRISPR/CasRx-based RNA targeting and proximity labeling to identify binding proteins of specific lncRNAs in native cellular context. Applying to the nuclear lncRNA XIST, CARPID reliably captured a list of its known interacting proteins. In addition, we also discovered multiple novel XIST-binding proteins, including a transcription initiation factor TAF15 and an ISWI chromatin remodeler SNF2L, which displayed distinct activities in X-inactivation. Furthermore, we generalized CARPID to explore binding proteins of a tumor-associated lncRNA DANCR and interestingly revealed an enrichment of proteins associated with extracellular exosomes, suggesting a pathway for its presence in circulation. Notably, CARPID can be harnessed to recover the labeled proteins from exosomes. The DANCR-dependent labeling of exosomal proteins were significantly reduced by downregulation of DANCR with active CasRx. We conclude that CARPID is a reliable and robust method to dissect the binding proteins of lncRNA inside living cells. Jingyu Li1,2, Wenkai Yi2, Xiaoxuan Zhu2, Ligang Fan2, Kui Ming Chan1,2, Jian Yan2, Liang Zhang1,2 1. Key Laboratory of Biochip Technology, Biotech and Health Centre, Shenzhen Research Institute of City University of Hong Kong, Shenzhen, China 2. Department of Biomedical Sciences, City University of Hong Kong, Hong Kong SAR, China

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