Signatures of RNA binding proteins globally coupled to effective microRNA target sites

Abstract

MicroRNAs (miRNAs) and small interfering RNAs (siRNAs), bound to Argonaute proteins (RISC), destabilize mRNAs through base-pairing with the mRNA. However, the gene expression changes after perturbations of these small RNAs are only partially explained by predicted miRNA/siRNA targeting. Targeting may be modulated by other mRNA sequence elements such as binding sites for the hundreds of RNA binding proteins (RNA-BPs) expressed in any cell, and this aspect has not been systematically explored. Across a panel of published experiments, we systematically investigated to what extent sequence motifs in 3' untranslated regions (UTRs) correlate with expression changes following transfection of small RNAs. The most significantly overrepresented motifs in down-regulated mRNAs are two novel U-rich motifs (URMs), UUUUAAA and UUUGUUU, recently discovered as binding sites for the ELAVL4 (also known as HuD) RNA-BP. Surprisingly, the most significantly overrepresented motif in up-regulated mRNAs is the heptanucleotide AU-rich element (ARE), UAUUUAU, which is known to affect mRNA stability via at least 20 different RNA-BPs. We show that destabilization mediated by the transfected miRNA is generally attenuated by ARE motifs and augmented by URM motifs. These ARE and URM signatures were confirmed in different types of published experiments covering eight different cell lines. Finally, we show that both ARE and URM motifs couple to presumed endogenous miRNA binding sites in mRNAs bound by Argonaute proteins. This is the first systematic investigation of 3' UTR motifs that globally couple to regulation by miRNAs and may potentially antagonize or cooperate with miRNA/siRNA regulation. Our results suggest that binding sites of miRNAs and RNA-BPs should be considered in combination when interpreting and predicting miRNA regulation in vivo.

Figure 1.

The ARE stability motif is overrepresented in genes up-regulated after miRNA transfection. (A) UAUUUAU overrepresentation in up-regulated 3′ UTRs after transfection of two different miRNAs in HeLa cells. The plot shows the UAUUUAU running sum (left to right) of word overrepresentation scores in the ranked list of 3′ UTRs (black line) and running sums from 500 random permutations of the UAUUUAU word scores (red lines, see Methods). miR-128 is included as it has the strongest UAUUUAU correlation (FDR < 1 × 10⁻⁷) among the 11 different miRNA transfections in HeLa cells, while miR-133a transfection has the weakest correlation (P < 0.03, FDR < 0.11). (B) The gene set defined by UAUUUAU overrepresentation at the 0.05 level comprised 448 genes expressed in HeLa cells, and this gene set was significantly up-regulated in the 11 HeLa experiments (P < 2.5 × 10⁻⁶⁰, Kolmogorov-Smirnov [KS] one-tailed test, standardized expression changes pooled for all 11 experiments). (C) For each of the seven time points following miR-124 transfection in HepG2 cells, the plot shows the significance level (−log₁₀ P-value, Kolmogorov-Smirnov one-tailed test) for down-regulation of miR-124 targets relative to all expressed genes (blue line) and the UAUUUAU word correlation Z-score in up-regulated genes (red line, numbers correspond to the rank of the correlation score, N = 21 504 words).

Figure 2.

ARE and URM motifs affect miRNA mediated repression. (A) Expression changes for the 11 HeLA miRNA transfection experiments were standardized and pooled. ARE genes are defined as genes with UAUUUAU overrepresentation at the P < 0.05 level. miRNA target genes are genes with a target site of the transfected miRNA in the given experiments. The plot shows that miRNA target genes with ARE stability motifs were significantly up-regulated compared with miRNA targets without ARE motifs (P < 1.3 × 10⁻⁸, Kolmogorov-Smirnov one-tailed test, pooled data). (B) The same analysis was carried out for endogenous miRNA target genes. Endogenous miRNA target genes are defined as genes with predicted conserved target sites for an endogenous miRNA and no target sites for the transfected miRNA mimic in the individual experiment. Endogenous miRNA target genes with ARE stability motifs were significantly up-regulated compared with endogenous miRNA targets without ARE motifs (P < 9.1 × 10⁻¹³, Kolmogorov-Smirnov one-tailed test). (C) Similar to A, but using genes with URM1 motif overrepresentation. miRNA target genes with URM1 overrepresentation were significantly down-regulated compared with genes with miRNA target sites alone (P < 2.5 × 10⁻⁹, Kolmogorov-Smirnov one-tailed test).

Figure 3.

Schematic of the effects of ARE and URM motifs in genes after miRNA transfections. (A) Effect on extent of down-regulation with transfected miRNA target site alone, the attenuation of down-regulation with ARE motifs in addition to the transfected target site, and the enhancement of down-regulation with URM motifs in addition to transfected target sites. Down-regulation is multiplicative, suggesting a synergistic mechanism of URM1 and transfected target sites. (B) Endogenous miRNA targets are more up-regulated after miRNA transfection if they also have ARE stability motifs. Up-regulation mediated by endogenous target sites and ARE motifs is multiplicative when they co-occur in a 3′ UTR, suggesting a synergistic mechanism.

Figure 4.

Functional consequences of ARE and URM motifs after miRNA transfections in HeLa cells, (A) Gene set enrichment analysis (GSEA) showing that genes with ARE motif overrepresentation are enriched for inflammatory pathways (INFLAMPATHWAY). (B) Genes up-regulated after miRNA transfections were enriched for cytokine-cytokine receptor interaction (HSA04060_CYTOKINE_CYTOKINE_RECEPTOR_INTERACTION). (C) Genes with overrepresentation of the URM1 motif were enriched for a manually curated gene set (CHESLER_HIGHEST_FOLD_RANGE_GENES) of neurologically relevant transcripts with high fold range between mouse inbred strains. (D) Genes down-regulated after miRNA transfections were also enriched for this gene set.