Supplemental Data for:
Lewis et al., Cell 120, pp. 15–20


Experimental Procedures and False-Positive Estimates
Algorithmic Description for TargetScanS
The TargetScanS algorithm is a simplified version of the TargetScan algorithm (Lewis et al., 2003) that searches multiple alignments to identify conserved W-C hexamer seed matches to the designated seed region of the miRNA (bases 2–7), flanked by either a W-C match to the m8 position of the miRNA or a conserved adenosine in the t1 position of the target, designated as the t1A anchor.
Identification of Conserved 3' Pairing
A modified version of the TargetScan algorithm was used for the identification of predicted target sites with conserved pairing to the 3' portion of the miRNA (Figure 2).  This algorithm proceeded by first finding matches to the miRNA seed that are W-C except at one position at which they have either a conserved G:U wobble or a conserved mismatched to the seed. This initial match was then extended with W-C pairing to m8 and by identifying the presence of a t1A anchor (W-C pairing to m1 was not scored). To identify pairing to the 3' portion of the miRNA, the miRNA and target site candidate that included the seed match and 15 mRNA bases upstream of this match were co-folded using subroutines imported from the RNAlib C program library of the Vienna RNA package (Hofacker et al., 1994), while incorporating constraints on the pairing of the miRNA seed. This routine was repeated using aligned sequences from each vertebrate genome. Predicted target sites were accepted in the 3'-pairing analysis if in each genome they contained a contiguous helix of at least six basepairs (allowing for a single G:U wobble), even if in the different genomes this helix involved different 3' residues of the miRNA.
MicroRNA Datasets
A set of 117 human miRNAs (Supplemental Table S1) representing 148 human miRNA genes with membership in 62 conserved vertebrate miRNA families was assembled using the Rfam miRNA registry ( and established criteria (Ambros et al., 2003). MicroRNA families were defined by grouping miRNAs that share a common conserved seed region spanning nucleotides 2–7 (although in any analysis involving m8 matches, miR-101 and miR-144 miRNAs, which have the same seed, were regarded as separate families because they differ at m8, bringing to 63 the total number of families for these analyses). One representative from each miRNA family was required to be conserved with no more than one mismatch to the sequence for mouse, rat, dog, and chicken sequences from the UCSC genome browser multiz-8-way whole-genome alignments (Karolchik et al., 2003; Blanchette et al., 2004). In a number of cases, a related miRNA from the chicken genome could not be found in the multiz-8-way alignments, and a chicken miRNA from the miRNA registry that satisfied aforementioned alignment criteria was used in its place. To account for documented 5' heterogeneity of miR-124 (Lagos-Quintana et al., 2002), two forms of miR-124 were included separately among the miRNA families: the longer, less frequently observed form contains an additional 5' U relative to the shorter form and is listed as miR-124u (Supplemental Tables S1, S2, and S3).
MicroRNA seed sequences corresponding to selected sets of families from the miRNA dataset were used in the analyses in Figures 1B–1G. These sets are listed in Supplemental Table S1 and described below:
Panels 1B, 1F, and 1G:  all 63 miRNAs families described above (62 in the case of Seed or Seed + t1A searches).
Panel 1C:  48 miRNA families representing only those sequences that correspond to seed regions (nucleotides 2–7) with no overlapping relationship with a shifted seed sequence of another miRNA family (from the same superfamily) were chosen to ensure the proper register of seed matches and conservation in the surrounding bases.
Panel 1D:  9 miRNA families corresponding to miRNA sequences that have a conserved m1 nucleotide other than U and do not have the same seed sequence as an miRNA with a U at m1.
Panel 1E:  36 miRNA families corresponding to miRNA sequences that have a conserved m9 nucleotide other than U and do not have the same seed sequence as an miRNA with a U at m9.
Genomic Datasets
The chromosomal coordinates of the 3' UTRs of all human genes from the “known genes” dataset (dated 7/2004) of the UCSC genome browser annotation database ( were used to define an initial dataset of human 3' UTR sequences. This set was augmented by taking the union of these regions with analogous regions defining the 3' UTRs of overlapping human Refseq mRNAs (Pruitt et al., 2003). The corresponding sequence coordinates were retrieved from the UCSC annotation database multiz-8-way multiple alignments (Blanchette et al., 2004), containing aligned orthologous sequences from recent assemblies of the mouse (mm5, 5/2004), rat (rn3, 6/2003), dog (canFam1, 7/2004), chicken (galGal2, 2/2004), Fugu (fr1, 8/2002), and zebrafish genomes (danRer1, 11/2003). When an annotated 3' UTR sequence overlapped an open reading frame, the overlap was masked to prevent contamination of the 3' UTR dataset with protein-coding sequences. Using the knownCanonical database from the UCSC genome browser, alternate isoforms of a common gene were identified. The longest 3' UTR among each set of multiple isoforms was chosen. The resulting dataset contained 17,850 aligned mammalian 3' UTRs (human/mouse/rat/dog) and 10,938 aligned vertebrate 3' UTRs (human/mouse/rat/dog/chicken). For a few genes, a longer aligned mammalian 3' UTR isoform was not conserved in chicken while a shorter  3' UTR isoform was conserved to chicken. These rare cases resulted in a specific vertebrate 3' UTR isoform not being included in the mammalian set. 
The 5' UTR and ORF datasets were compiled using genomic coordinates from the human RefSeq mRNA database of the UCSC genome browser to retrieve regions of the multiz-8-way alignments in a manner analogous to the construction of the 3' UTR datasets.  Isoforms of a common gene were identified after mapping the knownCanonical database to RefSeq. As before, the single longest sequence was chosen from each set of isoforms.  In addition, all ORF sequences were required to begin with a conserved start codon, and all protein-coding sequence was masked in the 5' UTR dataset. The resulting dataset of 5' UTRs contained 6623 sequences, and the resulting dataset of ORFs contained 11,830 sequences.
Density of Conservation in 3' UTRs
Analysis of Figure 1F:  For each 3' UTR in the dataset used in the 5-genome analysis, the number of conserved 7-mers was counted and a measure of the density of conservation in that 3' UTR was calculated by determining the average number of conserved 7-mers per 1000 nt. The 3' UTRs were sorted by this density measure and then assigned to bins such that each bin contained a sufficient number of 3' UTRs to give a total of ~8000 conserved 7-mers per bin. 
Analysis of Figure 1B (in islands):  The aligned 3' UTR sequences within 250 nt 5' and 3' of a conserved seed match were examined to determine a local density of conservation.  All target sites located in a ~500-nt region with fewer than a total of 50 conserved heptamers in the upstream and downstream windows were designated as occurring in islands of conservation. For cases where a 3' UTR boundary occurred within 250 nt, the number of conserved 7-mers per 1000 nt was calculated and those sites with a local density of less than 100 conserved 7-mers per 1000 nt were included in the islands of conservation set.
Generation of Control Sequences
Mononucleotide, dinucleotide, hexamer, heptamer, and octamer counts and frequencies were determined for all human 3' UTR sequences. Sets of control sequences were designed for each seed match sequence and each extended-match variant (SeedM + m8, SeedM + t1A, etc.) so as to preserve the expected frequency of random matching between miRNA seed sequences and complementary 3' UTR sequences. All hexamers, heptamers, heptamers, and octamers were examined to identify suitable control sequences for each miRNA seed (or augmented seed) that preserve (1) E(SM), the 1st order Markov probability of the seed match, and (2) O(SM), the observed count of seed matches in human UTRs within a total margin of  ±7.5%. As described in Lewis et al. (2003), for a miRNA seed match heptamer S1,S2,S3,S4,S5,S6,S7, E(SM) was equal to (PS1·PS1,S2·PS2,S3·PS3,S4·PS4,S5·PS5,S6·PS6,S7) where PS1 was the frequency of the nucleotide S1 and PSk,Sk+1 was the conditional frequency of the nucleotide Sk+1 given Sk at the previous position, determined by counting dinucleotides in the UTR sequences. Sequences corresponding to known miRNA seeds, as well as sequences known to function in mRNA processing, such as the polyadenylation signals AAUAAA and AUUAAA and the consensus RNA binding sequences of the puf protein family (White et al., 2001), were restricted from use in the control sets. All possible control sequences that met these criteria were assigned to each distinct miRNA seed sequence represented in the dataset. For each miRNA analyzed, an estimate of the false-positive predictions was calculated by averaging the results of each of its control sequences.  These averages were then summed to estimate the number of false positives for a set of miRNAs. A few miRNAs were assigned only five control sequences, but most had many more. For the analysis of target sites with pairing to the 3' end of the miRNA, control sequences were generated by simply merging each controlled seed sequence with the remaining ~13–16 nt region of the real miRNA that follows the seed.
For the analysis of miRNA target sites with a single G:U pair disrupting the seed match (Figure 2), control sequences were screened further so as to contain, on average, a similar number of instances of the 4-mer UGUA as the corresponding real miRNAs because this 4-mer is the core conserved element of the PUM2 binding site consensus (White et al., 2002). This extra measure was necessary for the G:U analysis due to the fact that the set of 6-mers corresponding to seed matches disrupted with a single G:U are enriched for the UGU 3-mer relative to 6-mers of comparable abundance in a single genome. The enrichment for UGU is a consequence of the enrichment for U’s and G’s obtained when specifying that seed:seed match duplexes must be disrupted by a G:U pair. 
To calculate the standard deviation of the number of mRNAs that are predicted to be targets of a single cohort of control sequences, in which a single cohort set consisted of one control sequence per real miRNA, a special procedure was devised that accounted for the varying number of control sequences assigned to each real miRNA in our set. The total number of cohort sets used was defined as N, which was equal to the maximum number of cohorts used for a single real miRNA in the set under consideration. The 1st listed control for each miRNA was assigned to cohort set 1, the 2nd to cohort set 2, … , the Nth to cohort set N. When considering cohort set n and a real miRNA with m control sequences in which m < n, the (n mod m)th control sequence was re-chosen to be included in the nth cohort set, thus enabling the construction of N total cohort sets.  For each cohort set, the number of predicted target mRNAs was determined, and the standard deviation of the mean was calculated. This single standard deviation value corresponds to the length of the error bars above and below the average noise level in the predicted targets plots (Figures 1B, 1D, 1E, 1G, 2A, and 2B).
Analysis of Gene Ontologies
Gene ontologies were assigned to human genes from the UCSC known genes database by crossreferencing with GO identifiers listed in the annotation database of the UCSC genome browser ( The Gene Ontology Consortium database (Harris et al., 2004) was retrieved from and biological process ontologies were compiled for all predicted target genes of the miRNAs (five-genome SeedM + m8M + t1A analysis) and the corresponding octamer control sequences. As in previous signal-to-noise calculations, the hits to GO categories were averaged to determine a noise estimate for an octamer control set and GO category.
Discussion Regarding Control Sequences for Estimating False Positives
Different approaches have been used to generate control sequences by which to estimate the number of false-positive miRNA target predictions. Our current approach is described above and resembles that which we used previously in mammalian predictions (Lewis et al., 2003). To evaluate the miRanda predictions, John et al. (2004) use an alternative approach to generate control sequences, wherein they shuffle (randomly permute) the miRNA. However, when analyzing vertebrate genomes, repeating a target prediction procedure with random shuffles is prone to underestimate the false positives, thereby overestimating the predictive accuracy of the algorithm (Lewis et al., 2003).
Pitfalls of using random shuffles to estimate the false positives can be illustrated with the miR-125 seed heptamer (CCCUGAG, 6-nt seed plus m8). This heptamer has 663 reverse-complement matches in human UTRs, whereas, on average, random shuffles have only 205 hits (Supplemental Table S4). It is unlikely that the difference of 458 hits represents the number of functional target sites for miR-125. Instead, this difference can be readily explained as an artifact of the shuffled sequences containing an oligonucleotide composition that differs from that of the miRNAs. For example, the miRNAs, like the UTRs and vertebrate genomes as a whole, contain few CG dinucleotides. Therefore (since CG is palindromic), random shuffles that create CG dinucleotides have far fewer hits to the UTRs than does the authentic seed heptamer (Supplemental Table S4). To avoid this artifact that would unduly favor the assessment of any algorithm that uses pairing or predicted duplex stability for prediction, it is better to pick controls sequences that match the relevant features of the authentic miRNAs, including compositional features (Lewis et al., 2003). For the miR-125 heptamer, the sequence CAGUGCC would be a more appropriate control than the typical shuffled derivative. The same principles can be used to generate control sequences that are the same length as the miRNAs (Lewis et al., 2003).
Comments on Overlapping Predictions
John et al. (2004) report that 46% of 400 target genes predicted by TargetScan (Lewis et al., 2003) overlapped with the targets predicted by miRanda. However, in most cases miRanda and TargetScan paired these UTRs to different miRNAs, and the actual overlap of predicted regulatory relationships was only 7%.
How might the 18% overlap of the miRanda predictions with the mammalian TargetScanS predictions be rationalized, given that a high fraction of miRanda predictions were estimated to be false positives? The set of miRanda predictions that overlap with TargetScanS predictions are enriched for highly conserved UTRs relative to the full set of 4-genome TargetScanS predictions. Having observed that TargetScanS is less accurate when applied to UTRs with higher levels of background conservation (Figure 1F), we would expect this overlapping set to have a signal:noise lower than the 2.2:1 signal:noise calculated for the set of more than 22,000 mammalian TargetScanS predictions. In addition to being enriched in lower-confidence TargetScanS predictions, this overlapping set would be expected to be enriched in higher-confidence miRanda predictions. This is because the overlap was determined using miRanda predictions for the subset of miRNAs used in the TargetScanS analysis, which were the more broadly conserved miRNAs and previously have been shown to yield higher signal:noise than do the less broadly conserved miRNAs (Lewis et al., 2003).
The Role of Evolutionary Conservation in miRNA Target Identification
An ultimate goal of miRNA target predictions will be to reliably identify authentic regulatory interactions without relying on conservation; this would find the regulatory interactions specific to each species. However, neither TargetScan nor any of the other target prediction methods are close to achieving this goal. In the meantime, we favor the approach of choosing the control sequences such that they give the same number of hits in a single genome as do the authentic miRNAs, and then relying on conservation of the sites in multiple genomes to distinguish the signal from the noise (e.g., Figure 1G).  Nonetheless, we recognize that this approach is conservative in that it will overestimate the noise to some extent, depending on both how well the algorithm predicts authentic targets in a single genome and the degree to which there has been selective pressure for transcripts to avoid miRNA complementarity. 
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Supplemental Figure S1.  An Illustration, Modeled after Figure 2 of John et al. (2004), Showing How Artifactual Signal above Noise Can Be Compounded when Requiring Multiple Sites per UTR
The height of each bar indicates the number of human transcripts containing a given number of predicted target sites. The 21-nt segments immediately upstream or downstream of miRNA hairpins were compiled as a set of fictitious miRNAs that will yield only false-positive predictions.  TargetScanS analysis was performed using these fictitious RNAs to generate a large set of predicted target sites (dark blue bars).  When using controlled shuffled miRNAs, this estimate matched the number of predictions from these fictitious miRNAs (black bars).  When using simple shuffles as the control sequences, the estimate of the noise was substantially lower (light blue bars), giving an errantly high signal above noise.  This artifact was compounded when asking for more than one conserved hit per UTR.
Supplemental Table S1. Supplemental Table S1.  Human microRNAs Used for Target Prediction
Supplemental Table S2. Supplemental Table S2. Predicted targets conserved in five vertebrates (Figure 1B)

Supplemental Table S3. Supplemental Table S3. Predicted Targets Conserved in Human, Mouse, Rat and Dog
Supplemental Table S4. Matches for the miR-125 Seed Heptamer and Its Shuffled Derivatives