RNA Immunoprecipitation and Sequencing of Isolated RNAs (RIP-SIR) Identifies Endogenous miRNA-Target Interactions
MicroRNAs (miRNAs) are crucial post-transcriptional regulators of gene expression in species ranging from plants to mammals. Unraveling the complex networks of miRNA-mediated regulation of gene expression requires the ability to identify the mRNA targets of individual miRNAs in native cellular contexts. Here we describe RIP-SIR, an unbiased genome-wide method for identifying interactions between endogenous miRNAs and their targets in almost any tissue or cell type. Overall design: Experimental methods for identifying the targets of individual miRNAs typically require expression of exogenous, tagged miRNAs which may perturb native stoichiometry, are limited to the study of cultured cells and cannot be used to monitor miRNA-mediated regulation during processes such as differentiation or disease progression. Additional methods such as Argonaute High-Throughput Sequencing of RNAs isolated by CrossLinking and ImmunoPrecipitation (Ago HITS-CLIP) and CrossLinking And Sequencing of Hybrids (CLASH), cannot match individual miRNAs to their targets or rely upon very low efficiency intramolecular ligation, respectively 9,10. To overcome the limitations of current methods for miRNA target identification, we developed RNA ImmunoPrecipitation and Sequencing of Isolated RNAs (RIP-SIR) and demonstrate that RIP-SIR identifys which miRNAs are bound to individual transcripts in lung tissues from both healthy mice and those bearing late-stage pulmonary adenocarcinomas.