Characterization of the miRNA turnover landscape and its regulation in Arabidopsis

The intracellular accumulation of microRNAs (miRNAs) is dynamically controlled at multiple levels, including transcription, processing, and degradation. However, the molecular mechanisms of miRNA turnover and active degradation remain understudied. Here, we delineated the global small RNA turnover landscape in Arabidopsis thaliana, using transcription inhibition combined with NaIO4 oxidative small RNA sequencing (OX-sRNA-seq), which specifically captures 2′-O-methylated small RNAs. We also employed a nontoxic pulse-chase approach based on metabolic RNA labeling to corroborate these results. Our results showed that although most miRNAs and small interfering RNAs (siRNAs) are highly stable, a subset of miRNAs undergo rapid turnover. In contrast, the majority of miRNA*s are short-lived. Reverse genetic analysis further demonstrated that ARGONAUTE 1 (AGO1) and HUA ENHANCER 1 (HEN1) act in maintaining miRNA stability. Intriguingly, we found that HAWAIIAN SKIRT (HWS), a pivotal player in target mimicry induced miRNA degradation, is crucial for the degradation of short-lived miRNAs. Plants utilize both transcription shutoff and HWS-mediated miR398 clearance during oxidative stress responses. Furthermore, HWS is required to maintain miR399 homeostasis under phosphate-sufficient conditions and accelerate its degradation after prolonged starvation. Together, our results shed light on the dynamics and mechanisms of miRNA stability in plants.