MicroRNAs are endogenous ∼23-nucleotide RNAs that can pair to sites in the messenger RNAs of protein-coding genes to downregulate the expression from these messages. MicroRNAs are known to influence the evolution and stability of many mRNAs, but their global impact on protein output had not been examined. Here we use quantitative mass spectrometry to measure the response of thousands of proteins after introducing microRNAs into cultured cells and after deleting mir-223 in mouse neutrophils. The identities of the responsive proteins indicate that targeting is primarily through seed-matched sites located within favourable predicted contexts in 3′ untranslated regions. Hundreds of genes were directly repressed, albeit each to a modest degree, by individual microRNAs. Although some targets were repressed without detectable changes in mRNA levels, those translationally repressed by more than a third also displayed detectable mRNA destabilization, and, for the more highly repressed targets, mRNA destabilization usually comprised the major component of repression. The impact of microRNAs on the proteome indicated that for most interactions microRNAs act as rheostats to make fine-scale adjustments to protein output.Large-scale approaches for studying the regulatory effects of microRNAs (miRNAs) have revealed important insights into target recognition and function. These approaches include computational analysis of the selective maintenance or avoidance of miRNA complementary sites during evolution [1][2][3][4][5][6][7][8] and experimental identification of messages destabilized or those preferentially associated with argonaute proteins in the presence of a miRNA [7][8][9][10][11][12][13][14][15] . Despite their utility, none of these approaches directly measures the influence of a miRNA on protein output, which is the most relevant readout of its regulatory effects. The influence of miRNAs on protein output has instead been limited to single-protein analyses, primarily immunoblotting and reporter assays, and a medium-size proteomics analysis with detection of 504 proteins 16 . ©2008 Macmillan Publishers Limited. All rights reservedCorrespondence and requests for materials should be addressed to S.P.G. (steven_gygi@hms.harvard.edu) or D.P.B (dbartel@wi.mit.edu).. * These authors contributed equally to this work. Author Contributions The order of listing of the first three authors is arbitrary. C.S. and F.C. performed the experimental work with cells and animals. J.V. performed the mass spectrometry and associated computational analysis. D.B. performed the computational analysis of targeting. All authors contributed to the design of the study and preparation of the manuscript. Author Information Array data and small RNA sequencing data were deposited in the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/) under accession number GSE12075. NIH Public Access Proteomic consequences of added miRNAsTo acquire data sufficient to investigate the effects of miRNA regulation on the proteome, we applied a quantitative-ma...

Most metazoan microRNAs (miRNAs) target many genes for repression, but the nematode lsy-6 miRNA is much less proficient. Here, we show that the low proficiency of lsy-6 can be recapitulated in HeLa cells and that miR-23 (a mammalian miRNA) also has low proficiency in these cells. Reporter results and array data both indicate two properties of these miRNAs that impart low proficiency: their weak predicted seed-pairing stability (SPS) and their high target-site abundance (TA). These two properties also explain differential propensities of small interfering RNAs (siRNAs) to repress unintended targets. Using these insights, we expand the TargetScan tool for quantitatively predicting miRNA regulation (and siRNA off-targeting) so as to model differential miRNA (siRNA) proficiencies, thereby improving prediction performance. Moreover, we propose that siRNAs designed to have both weaker SPS and higher TA will have fewer off-targets without compromised on-target activity.