MiR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction [I]

Identifying the interaction partners of non-coding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA-cross-linking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-cross-linking and Argonaute 2-immunopurification followed by streptavidin affinity-purification of probe-linked RNAs provided selectivity in the capture of targets, identified by deep-sequencing. MiR-CLIP with pre-miR-106a, a miR-17-5p family member, identified hundreds of putative targets in HeLa cells, many carrying conserved sequences complementary to the miRNA seed but also many that were not predicted computationally. MiR-106a overexpression experiments confirmed that miR-CLIP captured functional targets, including H19, a long-non-coding RNA that is expressed during skeletal muscle cell differentiation. We showed that miR-17-5p family members bind H19 in HeLa cells and myoblasts. During myoblast differentiation levels of H19, miR-17-5p family members and mRNA targets changed in a manner suggesting that H19 acts as a sponge for these miRNAs. Overall design: Two replicates of four cDNA libraries were submitted to deep sequencing: an RNA “Input” sample from RNA-7-transfected cells; a sample of the Ago2-immunopurified RNA from cells treated with transfection reagent (“Mock”); a sample of the Ago2-immunopurified RNA from RNA-7-treatment (“Ago2-IP”); and the sample from miR-CLIP-purified RNA (“miR-CLIP”).