systematic identification of mrnas recruited to argonaute 2 by specific micrornas and corresponding changes in transcript abundance系统标识的mrna招募argonaute 2由特定小分子核糖核酸和相应的转录丰度的变化.pdf

Systematic Identification of mRNAs Recruited to Argonaute 2 by Specific microRNAs and Corresponding Changes in Transcript Abundance 1. 2,3. 2 1,2 2,3 David G. Hendrickson , Daniel J. Hogan , Daniel Herschlag *, James E. Ferrell , Patrick O. Brown * 1 Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, California, United States of America, 2 Department of Biochemistry, Stanford University School of Medicine, Palo Alto, California, United States of America, 3 Howard Hughes Medical Institute, Stanford University School of Medicine, Palo Alto, California, United States of America Abstract microRNAs (miRNAs) are small non-coding RNAs that regulate mRNA stability and translation through the action of the RNAi-induced silencing complex (RISC). Our current understanding of miRNA function is inferred largely from studies of the effects of miRNAs on steady-state mRNA levels and from seed match conservation and context in putative targets. Here we have taken a more direct approach to these issues by comprehensively assessing the miRNAs and mRNAs that are physically associated with Argonaute 2 (Ago2), which is a core RISC component. We transfected HEK293T cells with epitope-tagged Ago2, immunopurified Ago2 together with any associated miRNAs and mRNAs, and quantitatively determined the levels of these RNAs by microarray analyses. We found that Ago2 immunopurified samples contained a representative repertoire of the cell’s miRNAs and a select subset of the cell’s total mRNAs. Transfection of the miRNAs miR-1 and miR-124 caused significant changes in the association of scores of mRNAs with Ago2. The mRNAs whose association with Ago2 in