miR-CLIP capture of a miRNA targetome uncovers a lincRNA H19-miR-106a interaction.

Abstract

Identifying the interaction partners of noncoding RNAs is essential for elucidating their functions. We have developed an approach, termed microRNA crosslinking and immunoprecipitation (miR-CLIP), using pre-miRNAs modified with psoralen and biotin to capture their targets in cells. Photo-crosslinking and Argonaute 2 immunopurification followed by streptavidin affinity purification of probe-linked RNAs provided selectivity in the capture of targets, which were 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 noncoding 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.