a, Schematic of neutrophil labelling and analysis. Haematopoietic progenitors were isolated from wild-type (WT) or mir-223^-/Y (KO) male mice and cultured in SILAC media containing granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) for six days. To enhance differentiation, SCF was withdrawn over the next 42 h. Mature neutrophils were mixed, and proteins were size-fractionated for quantitative MS analysis. mRNA was also collected from the cultures and directly from mice for expression profiling. b, mir-223 expression detected with RNA blots probing for miR-223. One blot analysed total RNA from sorted subpopulations of cells cultured in vitro (left, with sorting profiles shown at the far left). The other blot analysed total RNA from cells cultured in vitro for eight days and from neutrophils isolated directly from bone marrow (right). As a loading control, blots were re-probed for U6 small nuclear RNA. Below each blot are the relative expression levels, normalized using the loading control. Radio-labelled RNA markers (M) are also shown. c, Analysis of neutrophils isolated directly from mice (left) and those derived in vitro from haematopoietic precursors (right), monitoring the effects of miR-223 loss on messages with single miR-223 sites in their 3′ UTRs. Plotted is the fraction of messages that changed at least to the degree indicated on the x axis, otherwise as in Fig. 1c. d, The fraction of upregulated proteins deriving from messages with miR-223 7-8mer 3′-UTR sites, plotted as in Fig. 1b. e, The impact of deletion of mir-223 on neutrophil proteins, considering proteins from messages with single miR-223 sites in their 3′ UTRs, plotted as in Fig. 1c. f, Targeting efficacy in ORFs (n = 69) and 3′ UTRs (n = 50), plotted as in Fig. 1e. g, Efficacy of single 7-8mer sites and multiple sites, plotted as in f.

a, Protein and mRNA changes for quantified proteins deriving from messages with at least one 8mer 3′-UTR site (blue, n = 55) or at least one 7mer (orange, an additional 250 proteins). The least-squares best fit to the 8mer data are shown (blue line), as are reference lines (grey), which both have slope of 1.0. Vertical error bars indicate 25th and 75th percentiles for independent measurements of protein changes. Horizontal error bars indicate standard errors of mRNA changes from three biological replicates, one of which was also used for the SILAC experiments. b, Protein and mRNA changes for quantified proteins deriving from messages without 7-8mer 3′-UTR sites, plotted as in a. One of seven random cohorts is plotted here; the other six are in Supplementary Fig. 6. c, Distribution of the indicated reference-set mRNAs and quantified proteins with respect to mRNA expression, as indicated by the array signals from cultured neutrophils. d, Response of quantified proteins and their respective mRNAs to mir-223 deletion, considering those messages with 7-8mer 3′-UTR sites, grouped by mRNA expression.

a, Canonical miRNA seed-matched sites2. b, The fraction of repressed proteins deriving from messages with miR-124 3′-UTR sites (filled orange bar). At each repression cutoff, the number of repressed proteins from messages without 3′-UTR sites (indicated in the open bar) was used to calculate the additional fraction expected by chance to have a site (dashed line, with the corresponding number of repressed proteins indicated below the dashed line). Above the dashed line is the surplus number of repressed proteins deriving from messages with sites. c, Response of proteins from messages with single miR-124 3′-UTR sites. Plotted is the fraction of proteins that change at least to the degree indicated on the x axis. Proteins from messages with multiple 3′-UTR sites were not considered. 6mer sites that were part of larger sites were not included in the 6mer distribution, and 7mers that were part of 8mers were not included in the 7mer distributions. d, Efficacy of single 3′-UTR sites when pooling data from miR-124, miR-1 and miR-181 transfections, plotted as in c. e, ORF and 3′-UTR targeting efficacy. Plotted is the average change (± standard error) of protein and corresponding mRNA for quantified proteins from messages with at least one 8mer in the ORF (n = 83) or 3′ UTR (n = 87) corresponding to the transfected miRNA (excluding messages with sites in both ORF and 3′ UTR).

Analogous results were observed using the transfection data sets (Supplementary Fig. 8). a, Performance of programs that consider site conservation. Plotted is the average protein derepression (± standard error) of genes with ≥1 conserved or non-conserved 7-8mer 3′-UTR site (grey) and of genes predicted to be miR-223 targets. The number of quantified proteins in each set is in parenthesis. b, Recognition of an adenosine (A) opposite the first nucleotide of the miRNA. The cumulative plot of protein changes after miR-181 transfection compares proteins from messages with no seed-matched 3′-UTR site to those from messages with the indicated single 3′-UTR site. c, Relationship between the scores of predicted targets and protein derepression. Predictions corresponding to quantified proteins were divided into three equal-size bins according to the scores proposed to indicate the quality of the prediction or degree of repression. Statistically significant differences between the bottom and top third are indicated (asterisk, P < 0.01, Mann-Whitney U-test). d, Response of the top 29 predictions of each algorithm, plotted as in a. e, Performance of programs that do not consider site conservation, displayed as in a and c. Also shown is the response of quantified proteins from messages with only non-conserved 7-8mer 3′-UTR sites, binned by total context score.