Functional microRNA targeting without seed pairing
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hall-mhall98-phd-biology-2026-thesis .pdf
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Author(s)
Hall, Matthew H.
Advisor(s)
Bartel, David P.
Date Issued
February 2026
Publisher
Massachusetts Institute of Technology
Abstract
MicroRNAs (miRNAs) are short (~21–24-nt) regulatory non-coding RNAs that are loaded into Argonaute (AGO) proteins and guide those AGO proteins to bind partially complementary target sites in mRNAs. AGO–miRNA complex (AGO–miR) binding to those mRNAs results ultimately in accelerated mRNA degradation, causing post-transcriptional repression. Complementarity to the miRNA seed (miRNA nucleotides 2–7) is typically both necessary and sufficient for a target site to bind its cognate AGO–miR, but multiple studies had reported AGO–miR binding in vitro, or association in cells, with target sites that featured alternative “non-canonical” patterns of pairing to the miRNA. The equilibrium binding affinity of individual miRNA–target interactions is highly predictive of mRNA repression in cells, and so non-canonical sites that appeared to bind AGO–miR with high affinity were of particular interest. Foremost amongst these, long stretches of perfect complementarity to the miRNA 3′-region (miRNA nucleotide 9 through to the end of the miRNA) were found to bind some AGO–miRs with high affinity in unbiased high-throughput assays, but it was not clear whether these stretches of complementarity constituted a new type of “3′-only” miRNA target site or whether the miRNA seed was also involved in the binding. In this thesis I confirm that the observed high-affinity binding is indeed “3′-only”, involving only the miRNA 3′-region, and firmly establish 3′-only sites as a new type of miRNA target site that is highly effective at binding AGO–miRs in vitro, and at imparting post-transcriptional repression to site-containing reporter mRNAs in cells, at least for some miRNAs. This result was surprising given that it had been proposed that pairing to the miRNA seed both precedes and licenses pairing to the miRNA 3′-region. I therefore worked to characterize the biochemical mechanism of 3′-only site binding, which I show is distinct from traditional seedbased target engagement in that it displays slower association kinetics and release of the miRNA 3′-end from the pocket in AGO wherein it is typically anchored. I also show that as few as 2–3 seed region base pairs (termed a “microseed”) within ~15 nucleotides upstream provides a noticeable boost to 3′-only site affinity, efficacy, and association rate, possibly because it allows some molecules to associate via traditional seed-based mechanisms. Finally, I conduct an updated survey of the sets of endogenous target sites across the 3′ UTRome for hundreds of conserved human miRNAs that includes the two newly characterized site types: 3′-only sites and 3′+microseed sites, and work towards establishing the relative contribution of these site types to endogenous miRNA-mediated regulation, and the basis for the different ability of different miRNA sequences to bind their respective 3′-only sites.
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Massachusetts Institute of Technology. Department of Biology
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