Sequencing of Captive Target Transcripts Identifies the Network of Regulated Genes and Functions of Primate-Specific miR-522

• Pull-down of biotin-miRNA-bound RNAs without crosslinking identifies miRNA targets
• Sequencing of RNase-treated pulled-down miRNA targets identifies MREs
• miR-522 MREs do not obey the canonical 3′ UTR seed-pairing rules
• The oncomir miR-522 promotes detachment, invasion, and mesenchymal gene expression

SummaryIdentifying microRNA (miRNA)-regulated genes is key to understanding miRNA function. However, many miRNA recognition elements (MREs) do not follow canonical “seed” base-pairing rules, making identification of bona fide targets challenging. Here, we apply an unbiased sequencing-based systems approach to characterize miR-522, a member of the oncogenic primate-specific chromosome 19 miRNA cluster, highly expressed in poorly differentiated cancers. To identify miRNA targets, we sequenced full-length transcripts captured by a biotinylated miRNA mimic. Within these targets, mostly noncanonical MREs were identified by sequencing RNase-resistant fragments. miR-522 overexpression reduced mRNA, protein levels, and luciferase activity of >70% of a random list of candidate target genes and MREs. Bioinformatic analysis suggested that miR-522 regulates cell proliferation, detachment, migration, and epithelial-mesenchymal transition. miR-522 induces G1 cell-cycle arrest and causes cells to detach without anoikis, become invasive, and express mesenchymal genes. Thus, our method provides a simple but effective technique for identifying miRNA-regulated genes and biological function.

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