miRNA Sensor HuR Compartmentalizes Ago2-Uncoupled miRNAs to Lipid Droplets to Buffer miRNA Activity in Mammalian Cells

miRNA activity must be optimized to ensure target gene expression in mammalian cells in response to specific needs. How and when miRNA activity is regulated remains an open question in gene expression regulation, with limited information on how cellular machinery senses miRNA levels to regulate miRNA expression and activity. Because miRNAs are stable molecules that can be reversibly used, the existence of a miRNA-sensing mechanism in mammalian cells has been anticipated. With ectopic expression of miRNAs in mammalian cells, we found a dose-dependent miRNA buffering mechanism in which miRNA export and its storage on lipid droplets are essential for miRNA activity optimization in mammalian cells. We found that the miRNA-binding protein HuR, known for its role in miRNA export, has a dual function in mammalian cells. HuR uncouples miRNAs from Ago2 to facilitate association of lipid droplets with nonfunctional miRNAs, a process that gets augmented in cells with high lipid droplet content and restricted extracellular export. Thus, HuR acts as a miRNA sensor, and the optimal activity and abundance of HuR regulate miRNA storage on lipid droplets or its export to maintain cellular miRNA homeostasis, thereby preventing detrimental effects of an excess miRNA pool in mammalian cells. Thus, targeting miRNA export or lipid droplet association is an important strategy for buffering cellular miRNA levels. Importance To ensure that target gene expression in mammalian cells is appropriately regulated, miRNA activity needs to be carefully fine-tuned. Although miRNAs are stable and can be reused, the precise mechanisms by which their levels are sensed and regulated remain unclear. Our research uncovered a fascinating dose-dependent miRNA buffering process that involves export and storage of miRNAs on lipid droplets—essential steps for precise regulation. We also found that HuR, a protein that facilitates miRNA export, plays a key role by uncoupling miRNAs from Ago2 and promoting their association with lipid droplets in cells with abundant lipid droplets and limited extracellular export capacity. Overall, HuR acts as a miRNA sensor, helping to balance miRNA storage and export to maintain cellular homeostasis and prevent the harmful effects of miRNA overactivity.