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

• DOI: 10.1038/nsmb.1991
• Publication Date: 2011-01-23T18:32:04Z
• Authors: Anthony K L Leung; Amanda G Young; Arjun Bhutkar; Grace X Zheng; Andrew D Bosson; Cydney B Nielsen; Phillip A Sharp
• Keywords: Ribonuclease III; 0301 basic medicine; Binding Sites; Guanine; Base Sequence; Sequence Analysis, RNA; Eukaryotic Initiation Factor-2; High-Throughput Nucleotide Sequencing; Article; Cell Line; 3. Good health; DEAD-box RNA Helicases; Mice; MicroRNAs; 03 medical and health sciences; Gene Expression Regulation; Argonaute Proteins; Endoribonucleases; Animals; Embryonic Stem Cells; Gene Deletion; Protein Binding

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.