Figure 1

Methods for ascertaining miRNA targets. (A) In the HITS-CLIP method [11], RISC is pulled down using an antibody against AGO2. RNAse digestion then reduces the full length mRNA targets to short fragments that can be sequenced along with the miRNAs using high-throughput sequencing. This method uncovers RISC occupancy on individual transcripts and therefore captures the binding sites, but it cannot directly link them to a particular miRNA. This information must be inferred from the pool of miRNAs present. (B) In this study we employed a biotin pull-down method [13], in which a synthetic biotinylated miRNA is transfected into cells, integrates into RISC, and is then pulled down using streptavidin beads. The associated mRNAs are then profiled by microarray or RNAseq. This method does not reveal the exact miRNA binding sites, but it does preserve the relationship between the miRNA and its targets, allowing the exploration of closely related miRNA species. (C) We examined four shifted isomiRs and their canonical partners, which have different seed sites but share centered sites (defined as 11 bp starting at position 3, 4 or 5). miR-17-5p and its isomiR are illustrated here.