Sequence, structure, and affinity of miR-34a binding sites determine repression efficacy.

MicroRNAs (miRs) regulate gene expression post-transcriptionally by guiding Argonaute (AGO) proteins to target mRNAs. Efficiently predicting the repressive effects of miRNAs remains limited largely due to an incomplete understanding of how mRNA:miR structure affects function. Using EMSAs, structural probing, luciferase reporter assays, and transcriptome analysis, we investigated the structural, biophysical, and functional interaction between the human tumour suppressor miR-34a and 12 mRNA targets. Comparison of isolated mRNA:miRNA duplexes and those bound within functional AGO2 revealed that while the binary duplex largely predicts AGO2-associated affinity and structure, AGO2 bidirectionally modulates binding by attenuating strong interactions and stabilising weaker ones. Furthermore, we show that the impact of supplementary pairing is more pronounced in targets with shorter seeds compared to those with full-length seeds and confirm this effect in a transcriptome-wide analysis. Finally, we identified three structural groups of mRNA:miR-34a-AGO2 complexes, adopting either a symmetrical structure, or a bulge on the mRNA or miR side. miR-bulged complex repression was strongly linked to mRNA:miR affinity, whereas mRNA-bulged complexes showed no such correlation. Our results thus identify structural and biophysical characteristics of mRNA:miR duplexes that contribute to repression efficacy, revealing a hierarchy of seed type, structure, and affinity that determine repression efficiency.