Efficient 3′-pairing renders microRNA targeting less sensitive to mRNA seed accessibility

MicroRNAs (miRNAs) are short RNAs that post-transcriptionally regulate gene expression by binding to specific sites in mRNAs. Site recognition is primarily mediated by the seed region (nucleotides g2-g8 in the miRNA), but pairing beyond the seed (3 '-pairing) is important for some miRNA:target interactions. Here, we use SHAPE, luciferase reporter assays and transcriptomics analyses to study the combined effect of 3 '-pairing and secondary structures in mRNAs on repression efficiency. Using the interaction between miR-34a and its SIRT1 binding site as a model, we provide structural and functional evidence that 3 '-pairing can compensate for low seed-binding site accessibility, enabling repression of sites that would otherwise be ineffective. We show that miRNA 3 '-pairing regions can productively base-pair with nucleotides far upstream of the seed-binding site and that both hairpins and unstructured bulges within the target site are tolerated. We use SHAPE to show that sequences that overcome inaccessible seed-binding sites by strong 3 '-pairing adopt the predicted structures and corroborate the model using luciferase assays and high-throughput modelling of 8177 3 '-UTR targets for six miRNAs. Finally, we demonstrate that PHB2, a target of miR-141, is an inaccessible target rescued by efficient 3 '-pairing. We propose that these results could refine predictions of effective target sites. Graphical Abstract

Automated summary

This study investigates the combined effect of 3'-pairing and secondary structures in mRNAs on miRNA repression efficiency. The results show that 3'-pairing can compensate for low seed-binding site accessibility and enable repression of otherwise ineffective sites. The study also reveals that miRNA 3'-pairing regions can base-pair with nucleotides far upstream of the seed-binding site and tolerate both hairpins and unstructured bulges within the target site.