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bioRxiv · 10.1101/2025.03.16.643529

A unifying model for microRNA-guided silencing of messenger RNAs

Chatterjee, T.·Mandal, S.·Ray, S.·Johnson-Buck, A.·Walter, N. G.
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Abstract

Silencing by the miRNA-guided RNA induced silencing complex (miRISC) is dependent on Ago2-chaperoned base pairing between the miRNA 5' seed (5'S) and a complementary sequence in the 3' untranslated region of an mRNA. Prevailing mechanistic understanding posits that initial 5'S pairing can further allow functional base pair expansion into the 3' non-seed (3'NS), while functionally distinct non-canonical pairing was reported between only the 3'NS and the mRNA coding sequence. We developed single-molecule kinetics through equilibrium Poisson sampling (SiMKEPS) to measure highly precise binding and dissociation rate constants of varying-length target sequences to 5'S and 3'NS in a paradigmatic miRISC isolated from human cells, revealing distinct stable states of miRISC with mutually exclusive 5'S and 3'NS pairing. Our data suggest conformational rearrangements of the Ago2-bound miRNA that regulate alternative 5'S-and 3'NS-driven target recognition. The resulting model reconciles previously disparate observations and deepens our acumen for successfully marshaling RNA silencing therapies. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/643529v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1e92bb5org.highwire.dtl.DTLVardef@1348249org.highwire.dtl.DTLVardef@559fdborg.highwire.dtl.DTLVardef@1721b74_HPS_FORMAT_FIGEXP M_FIG C_FIG In brief: In this study, Chatterjee et al. developed a technique to characterize and identify multiple conformations of the RNA-induced silencing complex (RISC) present in human cells. Two distinct pathways of target recognition through different RISC conformations reconcile previously observed divergent findings on the microRNA (miRNA) binding mechanism to messenger RNA (mRNA). HighlightsO_LISiMKEPS is developed to precisely measure binding and dissociation of miRISC:mRNA C_LIO_LISingle human miRISC molecules exhibit mutually exclusive seed and non-seed pairing C_LIO_LISiMKEPS unveils conformational changes of single miRNAs bound to Ago2 C_LIO_LIResulting model reconciles previously divergent findings, supporting RNAi therapies C_LI

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Chatterjee, T., Mandal, S., Ray, S., Johnson-Buck, A., Walter, N. G.. 2025-03-17. A unifying model for microRNA-guided silencing of messenger RNAs. https://doi.org/10.1101/2025.03.16.643529

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