Identifying Argonaute binding sites in Caenorhabditis elegans using iCLIP

• Determining endogenous miRNA target sites has remained a challenge.
• CLIP-based methods enable the identification of endogenous miRNA targets.
• iCLIP provides increased sensitivity and resolution by isolating all cDNA products.
• We have adapted iCLIP for the isolation of ALG-1 binding sites in Caenorhabditis elegans.

The identification of endogenous targets remains an important challenge in understanding microRNA (miRNA) function. Past approaches using in silico methods and reporter constructs lack biological context that may enhance or inhibit target recognition. To address these limitations, several labs have utilized crosslinking and immunoprecipitation (CLIP) of Argonaute (Ago) proteins to identify miRNA targets. Recently, the Ule Lab introduced individual-nucleotide resolution CLIP (iCLIP) to increase the sensitivity of identifying protein–RNA interaction sites. Here we adapt the iCLIP protocol for use in Caenorhabditis elegans to identify endogenous sites targeted by the worm Argonaute (ALG-1) primarily responsible for miRNA function.