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

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Abstract
MicroRNAs bind Argonaute proteins, guiding silencing of target mRNAs. CLIP-seq has now been used to map microRNA targets bound by Ago2 in a Dicer-dependent fashion in mouse ES cells. The study reveals that certain sets of transcripts, such as TGF-β signaling pathway components, tend to be targeted, and also identifies a G-rich motif in targets that may modulate regulation. MicroRNAs (miRNAs) are 19–22-nucleotide noncoding RNAs that post-transcriptionally regulate mRNA targets. We have identified endogenous miRNA binding sites in mouse embryonic stem cells (mESCs), by performing photo-cross-linking immunoprecipitation using antibodies to Argonaute (Ago2) followed by deep sequencing of RNAs (CLIP-seq). 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 cross-linked to Ago2 in both 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.

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