Target mRNAs were shown to feed back on the microRNAs (miRNAs) that repress them: abundant cognate target sites accelerate DICER1-mediated pre-miRNA processing and AGO2 loading, increasing production of the mature miRNA (Bose and Bhattacharyya, 2016). The molecular transitions underlying this effect — a proposed 'scanning-and-jump' model in which target-site engagement triggers dissociation of AGO2-associated DICER1 and thereby raises DICER1 processivity — have never been directly tested, and the in vivo quantitative balance between this target-driven de novo biogenesis and the opposing target RNA-directed miRNA degradation (TDMD) pathway remains unmeasured, even though the TDMD machinery itself (the ZSWIM8-CUL3 ubiquitin ligase) is now molecularly understood and genetically dissociable. The endogenous prevalence, quantitative limits, and strand-selection consequences of target-driven miRNA biogenesis are likewise unresolved. The problem remains open overall, while two parts of the original 2016 question have been substantially resolved by later work: the mechanism of target-assisted passenger-strand ejection (cryo-electron microscopy structures, 2026) and the mechanism of TDMD (2020-2026).

Background

MicroRNAs (miRNAs) are ~22-nucleotide regulatory RNAs that guide Argonaute (AGO) proteins to complementary sites, predominantly in 3' untranslated regions (UTRs) of target mRNAs, causing translational repression and mRNA destabilization. In the canonical human pathway, primary miRNA transcripts are cropped in the nucleus, and the resulting precursor hairpins (pre-miRNAs) are exported to the cytoplasm, where the RNase III enzyme DICER1 cleaves them into ~22-bp duplexes. DICER1 physically and functionally couples pre-miRNA cleavage to loading of one strand (the guide) into Argonaute 2 (AGO2), forming the miRNA-induced silencing complex (miRNP/RISC); the discarded strand is called the passenger strand (miRNA*). 'Seed' pairing refers to the nearly invariant Watson-Crick pairing of miRNA positions 2-8, which anchors most target recognition, while more extensive 3' pairing increases complex stability. The DICER1-AGO2 assembly stage is sometimes called the miRNA loading complex (miRLC), and DICER1 acts as a multiple-turnover enzyme that serially loads many AGO2 molecules.

Canonically, information flows from miRNA to target. Bose and Bhattacharyya (2016) demonstrated the reverse for cognate pairs: increasing the amount of a target mRNA (e.g., CAT-1 mRNA resurging in refed Huh7 hepatoma cells, or reporter constructs with let-7a or miR-122 binding sites) raised the level and activity of the corresponding mature miRNA. In vitro reconstitution with recombinant DICER1 and AGO2 attributed the increase to enhanced DICER1-mediated pre-miRNA processing. To explain how a binding site on an mRNA could stimulate an enzyme acting on a pre-miRNA, the authors proposed a scanning-and-jump model: immediately after a loading cycle, DICER1 stays associated with the newly formed AGO2-miRNP; the complex scans the mRNA, and upon stable hybridization at a target site, DICER1 dissociates and jumps to a free AGO2 for another round, so that abundant target sites increase DICER1's effective processivity. The paper explicitly flagged what remained unresolved: the precise molecular transitions and any required cofactors behind the dissociation step; the impossibility, at the time, of cleanly separating this de novo biogenesis from TDMD because no TDMD inhibitors or markers existed; the unresolved mechanism of passenger-strand removal during AGO2 loading; and the unknown prevalence and physiological impact of the phenomenon across endogenous miRNA-target networks, including a speculated role in strand-bias control and developmental 'arm switching' (preferential maturation of the 5p versus 3p arm of a pre-miRNA).

Later work transformed two of these unknowns. First, TDMD — turnover of preformed mature miRNAs induced by highly or unusually complementary target RNAs — proved to be mediated by a cullin-RING ubiquitin ligase containing the substrate adaptor ZSWIM8, which ubiquitylates and proteasomally degrades AGO, exposing the miRNA to nucleases (Shi et al. 2020; Han et al. 2020); the biochemical mechanism of AGO binding and polyubiquitylation by the ZSWIM8-CUL3 complex has since been defined (Farnung et al. 2026), and ZSWIM8 loss-of-function now provides exactly the genetic dissociation tool whose absence the 2016 paper lamented. Second, cryo-electron microscopy (cryo-EM) structures of human AGO2 caught RISC-assembly intermediates in which target RNA, even a partially complementary one, actively displaces the passenger strand ('target-assisted passenger ejection', TAPE; Zhang et al. 2026), molecularly vindicating the proposal that both duplex strands can remain AGO2-associated until target hybridization ejects the passenger. In contrast, structural work on human DICER1 (e.g., the dicing-state DICER-pre-miRNA structure of Lee et al. 2023) has not captured DICER1 in complex with AGO2 or with a target RNA, and no study has directly tested the scanning-and-jump model, quantified the in vivo balance between target-driven biogenesis and TDMD, or systematically measured how widespread target-driven biogenesis is across endogenous networks.

Problem Statement

Three linked questions remain open from the discovery that target mRNAs drive biogenesis of their cognate miRNAs (Bose and Bhattacharyya 2016), updated for what later literature resolved:

  1. Molecular mechanism of target-enhanced DICER1 processivity. Identify the molecular transitions, conformational changes, and any required cofactors by which target-site engagement of the AGO2-miRNP accelerates dissociation of AGO2-associated DICER1, thereby increasing DICER1 processivity — i.e., directly test, refine, or replace the proposed scanning-and-jump model, including how seed pairing versus more extensive 3' pairing differentially contributes.

  2. In vivo balance between target-driven de novo miRNA biogenesis and target RNA-directed miRNA degradation (TDMD). For miRNA-target pairs in which both processes can operate, quantify the net effect of target abundance on mature miRNA steady state, now that ZSWIM8-mediated TDMD is mechanistically understood and genetically dissociable, and determine under what cellular contexts each opposing flux dominates.

  3. Endogenous prevalence, quantitative limits, and strand-selection consequences. Establish, across endogenous miRNA/target networks in diverse cell types and in vivo tissues, how widely target-driven miRNA biogenesis operates, what quantitative relationship exists between target-site abundance and cognate mature miRNA production, and whether target abundance biases guide-strand selection or drives developmental arm switching (the physiological residue of the passenger-strand question, whose molecular ejection mechanism was resolved by Zhang et al. 2026).

These three components are each posed on its own terms and jointly serve one objective: to determine whether, how, and how widely target RNAs regulate the production of the miRNAs that repress them, as a feedback layer of gene regulation.

References

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