Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs

MicroRNAs (miRNAs) are 19-22nt non-coding RNAs that post-transcriptionally regulate mRNA targets. To identify endogenous miRNA binding sites, we performed photo-crosslinking immunoprecipitation using Ago2 antibodies, followed by deep-sequencing of RNAs (CLIP-seq) in mouse embryonic stem cells (mESCs). We also performed CLIP-seq in Dicer(−/−) mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was miRNA-dependent. A significantly enriched motif, GCACUU, was identified only in wild-type mESCs in 3′ untranslated and coding regions. This motif matches the seed of a miRNA family that constitutes ∼68% of the mESC miRNA population. Unexpectedly, a G-rich motif was enriched in sequences crosslinked to Ago2 in the presence and absence of miRNAs. Expression analysis and reporter assays confirmed that the seed-related motif confers miRNA-directed regulation on host mRNAs and that the G-rich motif can modulate this regulation.