genome-wide identification of targets and function of individual micrornas in mouse embryonic stem cells全基因组的识别目标和个人小分子核糖核酸在小鼠胚胎干细胞的功能.pdfVIP

Genome-Wide Identification of Targets and Function of Individual MicroRNAs in Mouse Embryonic Stem Cells 1 1 1 1¤a ´ 2¤b Sophie A. Hanina , William Mifsud , Thomas A. Down , Katsuhiko Hayashi , Donal O’Carroll , 3 1 1 Kaiqin Lao , Eric A. Miska , M. Azim Surani * 1 Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, United Kingdom, 2 The Laboratory for Lymphocyte Signaling and the Laboratory of Molecular Immunology, The Rockefeller University, New York, New York, United States of America, 3 Molecular Cell Biology Division, Applied Biosystems, Foster City, California, United States of America Abstract Mouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1-null ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA–sequencing. In the absence of Dicer1, the RISC complexes are largely devoid