MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to serve as guides, directing binding to partially complementary sites in mRNAs, ultimately causing post-transcriptional repression. Complementarity to the miRNA seed (miRNA nucleotides 2-7) is typically necessary and sufficient for repression. Here, we investigate unusual sites with extensive complementarity to the miRNA 3' region (nucleotide 9 and onwards) but without complementarity to the seed. The top examples of these 3'-only sites bind as well as top canonical sites and impart similar repression, which can be further boosted by as few as 2-3 additional pairs to the miRNA seed. Despite these similarities, 3'-only sites have slower association and dissociation rates than seed-matched sites. They also impart different conformations to bound AGO-miRNA complexes than do seed-matched sites, and individual miRNAs differ substantially with respect to how well they bind their respective 3'-only sites. Thus, pairing to the seed is not always required for binding and repression, or for a target to gain access to the 3' region of the guide. For miRNAs that recognize 3'-only sites, those sites are estimated to constitute <1% of the set of endogenous target sites, a proportion resembling that of other rare but functional site types such as 3'-compensatory sites.
© The Author(s) 2025. Published by Oxford University Press.