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An extensive forward genetic screen identifies the HWS-AGO1 axis as pivotal for target mimicry-mediated miRNA degradation in Arabidopsis

Created on 11 Nov 2025

Authors

Yang, L., Liu, M., Guo, M., Fan, Y., Wang, Y., Kang, G., Li, N., Fang, Y., Mei, J., Zhang, X., Yang, J., Ren, G.

Abstract

Target RNAs have emerged as key regulators of miRNA stability, thereby influencing development and physiological processes in both plants and animals. The F-box protein HAWAIIAN SKIRT (HWS) has been characterized as a crucial factor in targeted miRNA degradation in plants (pTMD), a process triggered by a specific type of target RNA called target mimicry. However, the precise mechanism by which HWS functions is still poorly understood. We previously established a genetic reporter system based on Short Tandem Target Mimicry of miR160 (STTM160) triggered miR160 degradation, resulting in pleiotropic developmental defects that are easy to monitor. Here, through an extensive forward genetic screen, we identified fourteen additional hws alleles that near-completely restored STTM160-induced developmental defects, highlighting a central role for HWS in pTMD. Intriguingly, we discovered two adjacent amino acid substitutions (R421K and G422D) in the PAZ domain of AGO1 that significantly suppressed the STTM160 phenotype. Similar to HWS dysfunction, the AGO1 R421K substitution caused a significant retention of target mimicry RNAs in AGO1 immunoprecipitates, suggesting that the R421 residue of AGO1 may cooperate with HWS for the clearance of AGO1-miRNA-target mimicry complexes. Although the R/G mutations only impacted a small set of endogenous miRNA levels, they were sufficient to rescue the developmental defects caused by HWS overexpression (HWS-OE), suggesting that the developmental abnormalities caused by HWS-OE largely depend on the AGO1-miRNA pathway. Taken together, our findings provide a solid genetic evidence for the coordination of HWS and AGO1 in pTMD.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 11 Nov 2025.

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