Identifying mRNA sequence elements for target recognition by human Argonaute proteins

Abstract

It is commonly known that mammalian microRNAs (miRNAs) guide the RNA-induced silencing complex (RISC) to target mRNAs through the seed-pairing rule. However, recent experiments that coimmunoprecipitate the Argonaute proteins (AGOs), the central catalytic component of RISC, have consistently revealed extensive AGO-associated mRNAs that lack seed complementarity with miRNAs. We herein test the hypothesis that AGO has its own binding preference within target mRNAs, independent of guide miRNAs. By systematically analyzing the data from in vivo cross-linking experiments with human AGOs, we have identified a structurally accessible and evolutionarily conserved region (∼10 nucleotides in length) that alone can accurately predict AGO-mRNA associations, independent of the presence of miRNA binding sites. Within this region, we further identified an enriched motif that was replicable on independent AGO-immunoprecipitation data sets. We used RNAcompete to enumerate the RNA-binding preference of human AGO2 to all possible 7-mer RNA sequences and validated the AGO motif in vitro. These findings reveal a novel function of AGOs as sequence-specific RNA-binding proteins, which may aid miRNAs in recognizing their targets with high specificity.

Figure 1.

AUC scores for the blind test of predicting AGO-associated sequences that do not have (A) or do have (B) miRNA binding sites. AUC scores were computed using sequence alone (solid curve) or using the dinucleotide composition within the 41-nt regions (dashed curve). The thin dashed line is a reference for a lack of predictive power.

Figure 2.

Dissecting sequence features underlying AGO target recognition for RNA fragments that have or do not have miRNA binding sites. (A,B) (Blue curve) AUC for predicting AGO binding using a 15-nt window on the independent test set sliding over the 81-nt sequences. Structural accessibility of each nucleotide is shown at the bottom (above the x-axis), where green, red, and blue represent unchanged, elevated, or reduced accessibility in comparison with the negative samples (false discovery rate <0.01). Statistical significance was determined by the paired Wilcoxon rank sum test between the positive and negative samples. (C,D) The discriminative power for the most defining regions (left column, corresponding to the peaks on the blue curves), the most accessible regions (middle column), and the subregions of the most defining regions after the crosslinking sites (right column). (E,F) Seventeen-way phastCons scores (y-axis indicates cross-species evolutionary conservation) of each nucleotide for an RNA fragment without (E) and with (F) miRNA binding sites. It is clear that AGO-associated sequences (the positive samples in red) tend to be more conserved than the negative samples (in blue). The highly accessible regions immediately flank the peaks, indicating elevated conservation for these regions of high accessibility. Error bars, 1 SEM.

Figure 3.

AGO recognizes its targets in a sequence-specific manner within the highly accessible region upstream of the crosslinking site. (A) The mRNA fragments that are depleted of miRNA binding sites have an overrepresented sequence motif within the ∼10-nt accessible regions. (B) Discriminative power of the motif among three independent test sets. (Left) A subset of the AGO-bound mRNAs that was not used in deriving the motif; note these mRNAs were depleted of miRNA binding sites. (Middle) The same data set after removing sequences with at least one 6-mer match to any miRNA seed region. (Right) AGO-bound mRNA sequences that are highly enriched for miRNA binding sites. Overrepresentation of the motif in each data set was assessed by the sum of the position-specific scoring matrix (PSSM) scores for its letters. The more positive scores indicate a greater enrichment for the sequences resembling the motif, while the negative scores indicate the depletion of this motif. (C) Discriminative power of the motif among the AGO-bound sequences in coding regions with (site-containing) and without (non-site-containing) downstream miRNA binding sites. (D) Nine-nucleotide sliding window scan for the AGO-bound sequences that were not used to derive the AGO motif. These sequences were centered at the crosslinking site at position 41. The x-axis is the starting position of the sliding window, and the y-axis is the PSSM score for the AGO motif inside each sliding window. The motif is strongly favored in the ∼10 nt immediate upstream (x = 32) of the crosslinking sites (x = 41) and is disfavored in most other regions (receiving negative PSSM scores). Error bars, 1 SEM.

Figure 4.

Significant correlation between RNAcompete scores and PSSM scores for the identified motif in replicate 1 (A) and replicate 2 (B). In both panels, the higher RNAcompete scores indicate greater binding specificities of AGO2 measured from the assay. Similarly, higher PSSM scores represent a greater resemblance of the identified motif. It is clear that sequences with high similarity with the identified motif tend to have a higher binding specificity of human AGO2.

Figure 5.

Weakly expressed miRNAs require a strong AGO motif (P = 5.7 × 10⁻³, Wilcoxon rank sum test). Motif strength was quantified by their PSSM scores in the motif region.

Figure 6.

In vitro validation for a previously reported AGO-associated motif. (A) The previously reported G-rich motif was able to be replicated using MEME from the original data (Dicer^−/− mutant from mouse embryonic cells, two biological replicates, Dicer^−/− replication 1 and 2). (B) The G-rich motifs received the lowest RNAcompete scores for human AGO2 (sharing ∼99.1% protein sequence similarity with mouse) in comparison with all probes on the RNAcompete array and also with the AGO motif in this study. (*) P < 1 × 10⁻⁵⁰, Wilcoxon rank sum test.