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 e ciency. Here we describe a CRISPR-Assisted RNA-Protein Interaction Detection method (CARPID), which leverages CRISPR/CasRx-based RNA targeting and proximity labeling, to rapidly capture binding proteins of speci c lncRNAs in their native cellular context followed by LC-MS/MS identi cation. Applied to a variety of lncRNAs of different lengths and subcellular localizations, CARPID is proven to be a reliable and robust tool to discover the binding proteins of lncRNAs inside living cells.

The recent discovery of the cancer-associated E76K mutation in histone H2B (H2BE76-to-K) in several types of cancers revealed a new class of oncohistone. H2BE76K weakens the stability of histone octamers, alters gene expression, and promotes colony formation. However, the mechanism linking the H2BE76K mutation to cancer development remains largely unknown. In this study, we knock in the H2BE76K mutation in MDA-MB-231 breast cancer cells using CRISPR/Cas9 and show that the E76K mutant histone H2B preferentially localizes to genic regions. Interestingly, genes upregulated in the H2BE76K mutant cells are enriched for the E76K mutant H2B and are involved in cell adhesion and proliferation pathways. We focused on one H2BE76K target gene, ADAM19 (a disintegrin and metalloproteinase-domain-containing protein 19), a gene highly expressed in various human cancers including breast invasive carcinoma, and demonstrate that H2BE76K directly promotes ADAM19 transcription by facilitating efficient transcription along the gene body. ADAM19 depletion reduced the colony formation ability of the H2BE76K mutant cells, whereas wild-type MDA-MB-231 cells overexpressing ADAM19 mimics the colony formation phenotype of the H2BE76K mutant cells. Collectively, our data demonstrate the mechanism by which H2BE76K deregulates the expression of genes that control oncogenic properties through a combined effect of its specific genomic localization and nucleosome destabilization effect.

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. Here we describe a CRISPR-Assisted RNA-Protein Interaction Detection method (CARPID), which leverages CRISPR/CasRx-based RNA targeting and proximity labeling, to rapidly capture binding proteins of specific lncRNAs in their native cellular context followed by LC-MS/MS identification. Applied to a variety of lncRNAs of different lengths and subcellular localizations, CARPID is proven to be a reliable and robust tool to discover the binding proteins of lncRNAs inside living cells.