Optimizing lncRNA-miRNA interaction analysis: Modified crosslinking and immunoprecipitation (M-CLIP) assay

Defining lncRNA-miRNA interactions is critical for understanding their roles in cellular signaling and cancer biology. Capturing these interactions is challenging due to the inherent instability of RNAs. Our study focuses on the long non-coding RNA (lncRNA) UCA1, exploring its role in ovarian cancer progression through interactions with microRNAs (miRNAs). We hypothesized that UCA1 acts as a competing endogenous RNA (ceRNA), sequestering let-7 miRNAs to modulate the expression of let-7 targets, thereby driving cancer progression. Typically, miRNAs associate with ribonucleoprotein complexes that include Ago2 protein, pivotal in mediating miRNA activity and stability. Analyzing these complexes has proven effective in identifying lncRNAs and their miRNA partners. Inspired by previous RNA-protein crosslinking methodologies, we developed the Modified Crosslinking and Immunoprecipitation (M-CLIP) assay to capture UCA1-let-7 miRNA interactions through immunoprecipitation of Ago2, followed by qRT-PCR to detect the bound UCA1 and its associated let-7 miRNAs. This method includes:•Formaldehyde-based crosslinking followed by cell lysis•Immunoprecipitation and isolation of RNAs bound to bait proteins•Characterization of bound lncRNA and target miRNAsOur findings demonstrate the efficacy of the M-CLIP assay in identifying UCA1-let-7 interactions, providing a robust tool to elucidate how UCA1 and similar lncRNAs influence cancer progression through miRNA sequestration. The graphical abstract summarizes the workflow of M-CLIP assay to analyze the lncRNAs and their miRNA partners. Cells were harvested, protein-RNA complexes were crosslinked with formaldehyde treatment, cells were lysed, immunoprecipitated with Ago2 and Sepharose beads, Ago2-bound RNAs were eluted along with reversal of crosslinking, followed by RNA isolation, reverse transcription of RNA and qRT-PCR for lncRNAs and miRNAs. [Display omitted]