Figure 1. Inefficacy of recently reported non-canonical sites.

(A) Response of mRNAs to the loss of miRNAs, comparing mRNAs that contain either a canonical or nucleation-bulge site to miR-430 to those that do not contain a miR-430 site. Plotted are cumulative distributions of mRNA fold changes observed when comparing embryos that lack miRNAs (MZDicer) to those that have miRNAs (WT), focusing on mRNAs possessing a single site of the indicated type in their 3′ UTR. Similarity of site-containing distributions to the no-site distribution was tested (one-sided Kolmogorov–Smirnov [K–S] test, P values); the number of mRNAs analyzed in each category is listed in parentheses. See also Figure 1—figure supplement 1C and Figure 1—figure supplement 4A. (B and C) Response of mRNAs to the loss of miR-155, focusing on mRNAs that contain either a single canonical or ≥1 CLIP-supported non-canonical site to miR-155. These panels are as in (A), but compare fold changes for mRNAs with the indicated site type following genetic ablation of mir-155 in either T cells (B) or T_h1 cells (C). See also Figure 1—figure supplement 2. (D and E) Response of mRNAs to the knockdown of miR-92a, focusing on mRNAs that contain either a single canonical or ≥1 CLASH-identified non-canonical site to miR-92a. These panels are as in (A), except CLASH-supported non-canonical sites were the same as those defined previously (Helwak et al., 2013) and thus were permitted to reside in any region of the mature mRNA, and these panels compare fold changes for mRNAs with the indicated site type following either knockdown of miR-92a (D) or combined knockdown of miR-92a and 24 other miRNAs (E) in HEK293 cells. See also Figure 1—figure supplement 3A,B. (F) As in (D), but focusing on mRNAs that contain ≥1 chimera-identified site. See also Figure 1—figure supplement 3C–E and Figure 1—figure supplement 4B. (G) Response of mRNAs to the transfection of 16 miRNAs, focusing on mRNAs that contain either a canonical or MIRZA-predicted non-canonical site. This panel is as in (A), but compares the fold changes for mRNAs with the indicated site type after introducing miRNAs, aggregating results from 16 individual transfection datasets. Fold changes are plotted for the top 100 non-canonical predictions for each of 16 miRNAs compiled either before (MIRZA, top 100) or after (MIRZA, no 6mers) removing mRNAs containing 6mer or offset-6mer 3′-UTR sites. (H) Response of mRNAs to a transfection of miR-522, focusing on mRNAs that contain either a single canonical or ≥1 IMPACT-seq–supported non-canonical site to miR-522. These panels are as in (A), except IMPACT-seq–supported non-canonical sites were the same as those defined previously (Tan et al., 2014) and thus were permitted in any region of the mature mRNA. (I) Response of ribosomes to the loss of miR-155, focusing on mRNAs that contain either a single canonical or ≥1 CLIP-supported non-canonical site to miR-155. This panel is as in (B and C) but compares the response of mRNAs using ribosome-footprint profiling (Eichhorn et al., 2014) after genetic ablation of mir-155 in B cells. Ribosome-footprint profiling captures changes in both mRNA stability and translational efficiency through the high-throughput sequencing of ribosome-protected mRNA fragments (RPFs).

DOI: http://dx.doi.org/10.7554/eLife.05005.003

Figure 1—figure supplement 1. Inefficacy of nucleation-bulge sites.

(A and B) These panels are as in Figure 1A but compare the response of cognate site-containing mRNAs in a compendium of either 11 miRNA transfection datasets (A) or 74 sRNA transfection datasets (B). The datasets were pre-processed (Figure 3) and are provided in Supplementary file 1. (C) This panel is as in Figure 1A but compares the response of mRNAs in MZDicer embryos in which miR-430 has been injected. (D–F) These panels are as in Figure 1A but compare the response of mRNAs with the indicated miR-124 site types after transfecting miR-124 into either HEK293 cells (D), HeLa cells (E), or Huh7 cells (F).

DOI: http://dx.doi.org/10.7554/eLife.05005.004

Figure 1—figure supplement 2. Inefficacy of CLIP-supported non-canonical miR-155 sites.

(A and B) These panels are as in Figure 1B but compare the response of mRNAs after genetic ablation of miR-155 in Type 2 helper T cells (T_h2, A) or B cells (B).

DOI: http://dx.doi.org/10.7554/eLife.05005.005

Figure 1—figure supplement 3. Inefficacy of CLASH- and chimera-supported non-canonical sites.

(A–D) These panels are as in Figure 1D but compare the response of mRNAs with sites cognate to any one of four miRNA families (miR-15/16, miR-19, miR-17/20/93/106, or miR-25/92), for either all CLASH-supported targets (A), mRNAs with CLASH-supported 3′-UTR sites (B), all chimera-supported targets (C), or mRNAs with chimera-supported 3′-UTR sites (D). These four miRNA families were chosen because their predicted targets were the most responsive to knockdown of the 25 miRNAs. p values reflect the median p value (as evaluated by a K–S test) across 100 trials in which a no-site control cohort with matched 3′-UTR lengths was chosen for each site-containing distribution. Length-matched no-site controls were required for this analysis because longer 3′ UTRs had a greater chance of containing additional sites to at least one of the many miRNAs that were knocked down, and thus had a greater chance of being derepressed as a result of interactions otherwise not considered in the analysis. To populate each control cohort, 500 different no-site mRNAs were chosen, considering the 3′-UTR length of each site-containing mRNA and selecting (without replacement) control mRNAs from among the 10 no-site mRNAs with the most similar 3′-UTR lengths. Shown is the response of a control cohort for mRNAs containing non-canonical sites. mRNAs with 3′ UTRs >2000 nt were excluded from the analysis because so many of the 3′ UTRs >2000 nt had a site to at least one of the four miRNA families, making it impossible to select appropriate length-matched controls. (E) This panel is as in Figure 1F but compares the response of mRNAs with the indicated miR-302 site types after knocking down miR-302/367 in hESCs.

DOI: http://dx.doi.org/10.7554/eLife.05005.006

Figure 1—figure supplement 4. Inefficacy of non-canonical sites in mediating translational repression.

(A) This panel is as in Figure 1A but compares the response of mRNAs using ribosome footprint profiling (Bazzini et al., 2012), which captures changes in both mRNA stability and translational efficiency through the high-throughput sequencing of ribosome-protected mRNA fragments (RPFs). (B) This panel is as in (Figure 1I) but compares protein fold changes for chimera-supported targets, as evaluated by pulsed SILAC (Selbach et al., 2008) after transfection of miR-155 in HeLa cells.

DOI: http://dx.doi.org/10.7554/eLife.05005.007

Figure 1—figure supplement 5. Re-evaluating conservation of chimera-supported non-canonical sites.

(A) Conservation of chimera-supported non-canonical sites detected in an analysis modeled after that of Grosswendt et al. (2014) but modified to control for background conservation. Plotted for the indicated miRNAs is the average conservation of chimera-supported non-canonical sites, as measured by branch-length score (BLS), compared to the average conservation of 100 equally sized cohorts of controls; error bars, standard deviation of cohort averages; **, p < 0.01; *, p < 0.05, one-sided Z test. We considered chimera-supported non-canonical sites that mapped within 3′ UTRs and contained a single mismatch to the 6 nt seed of the miRNA. This set of sites mirrored that analyzed previously (Grosswendt et al., 2014), and excluded offset 6mers, which as a class was already known to mediate repression and exhibit preferential conservation (Friedman et al., 2009). Cohorts of control sites were generated such that for each chimera-supported site, each control cohort contained a single example of the identical 6 nt motif that was present in the indicated region (either an AGO cluster or 3′ UTR) but not supported by chimeric reads. To control for local background conservation and thereby avoid treating sites within slowly evolving 3′ UTRs the same as those within rapidly evolving 3′ UTRs, we used the binning procedure developed for calculating P_CT scores (Friedman et al., 2009); 3′ UTRs were partitioned into 10 conservation bins (based on the median BLS of the nucleotides of the human sequence), and control sites were randomly selected (with replacement) from 3′ UTRs in the same bin as the actual site. Control AGO clusters were collected as was done previously (Grosswendt et al., 2014), using genome-wide data downloaded from clipz.unibas.ch and derived from multiple AGO PAR-CLIP experiments performed in HEK293 cells (Kishore et al., 2011). The union of AGO clusters for all experiments was computed and filtered for overlap with Ensembl-annotated 3′ UTRs, using the ‘merge’ and ‘intersectBED’ utilities, respectively, found in BEDTools v2.20.1 (parameter ‘-s’) (Quinlan and Hall, 2010). (B) Attribution of the conservation signal to the confounding effects of conserved regions. Considered are 1443 non-canonical chimera-supported sites selected as in (A) but including sites of all miRNA families. For each chimera-supported site, a z score was generated using the distribution of BLSs for 100 control sites chosen as in panel (A) from either AGO clusters or 3′ UTRs, as indicated. Each z score reflected how the conservation of the actual site differed from that of its controls. Compared are cumulative distributions of the z scores for sites of broadly conserved miRNAs and those of less conserved miRNAs, using the previously defined sets of broadly and less conserved miRNAs (Friedman et al., 2009). If the chimera-supported non-canonical sites were preferentially conserved because of their function in mediating repression, then sites of broadly conserved miRNAs would be expected to have a right-shifted distribution compared to sites of less conserved miRNAs. However, no significant difference was discerned between each pair of z-score distributions. The remainder of this legend outlines the rationale for the analysis of this panel. One way to reconcile the conservation signal observed in panel (A) with our conclusion that a large majority if not all of these sites bind miRNA but do not mediate repression is to consider the potentially confounding biochemical properties of conserved regions, which are illustrated by the observation that artificial siRNAs preferentially target sites that are evolutionarily conserved over those that are not (Nielsen et al., 2007). Because these siRNAs are not natural (and do not share a seed with conserved miRNAs) the evolutionary conservation of these preferred sites could not have arisen because they function to mediate sRNA-guided repression. Instead, some other function of these 3′-UTR regions, such as greater accessibility to RNA-binding factors, must explain their preferential conservation and also endow them with properties that favor sRNA binding (Nielsen et al., 2007). To examine whether confounding properties of conserved 3′-UTR regions might similarly explain the elevated conservation of chimera-supported sites, we compared the z scores for sites bound by broadly conserved miRNAs (miRNAs in families conserved beyond mammals, as listed in TargetScan7) with those bound by less conserved miRNAs. MicroRNAs conserved among mammals but not more broadly were grouped with the less conserved miRNAs because canonical 6mer and 7mer sites to these miRNAs have no conservation signal above background, presumably because these miRNAs have not been present long enough for the number of preferentially conserved 6mer and 7mer sites to rise above the background (Friedman et al., 2009); we reasoned that the same would be true of non-canonical sites, to the extent that any are preferentially conserved. If the conservation signal observed in panel (A) were related to miRNA binding, we would have expected a difference between the scores for the sites of broadly and less conserved miRNAs. The lack of a significant difference supports the idea that chimera-supported non-canonical sites tend to be conserved for the same reason that functional sites to artificial siRNAs tend to be conserved.

DOI: http://dx.doi.org/10.7554/eLife.05005.008