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

Abstract

MicroRNAs and 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 small RNA targeting. MicroRNA/siRNA targeting may be modulated by other mRNA sequence elements such as binding sites for the hundreds of RNA binding proteins expressed in any cell. This aspect of small RNA regulation has not yet been systematically explored. Across a panel of published experiments using rigorous computational methods, we systematically investigated to what extent sequence motifs (words) in 3' UTRs correlate with expression changes following transfection of small RNAs. We discover hundreds of motifs having significant recurrent correlation with up or down-regulation in all transfection experiments. 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 HuD RNA binding protein. 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 twenty different ARE binding proteins. 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 microRNA binding sites in mRNAs bound by Argonaute proteins. This is the first systematic investigation of 3'UTR motifs which may be globally coupled to regulation by microRNAs and potentially antagonise or cooperate with microRNA/siRNA regulation. Our results suggest that microRNA and siRNA binding sites should be considered in combination with binding sites of RNA binding proteins when interpreting and predicting effects of these small RNAs in-vivo.