Argonaute-bound microRNAs silence mRNA expression in a dynamic and regulated manner to control organismal development, physiology, and disease. We employed metabolic small RNA sequencing for a comprehensive view on intracellular microRNA kinetics in Drosophila. Based on absolute rate of biogenesis and decay, microRNAs rank among the fastest produced and longest-lived cellular transcripts, disposing up to 10⁵ copies per cell at steady-state. Mature microRNAs are produced within minutes, revealing tight intracellular coupling of biogenesis that is selectively disrupted by pre-miRNA-uridylation. Control over Argonaute protein homeostasis generates a kinetic bottleneck that cooperates with non-coding RNA surveillance to ensure faithful microRNA loading. Finally, regulated small RNA decay enables the selective rapid turnover of Ago1-bound microRNAs, but not of Ago2-bound small interfering RNAs (siRNAs), reflecting key differences in the robustness of small RNA silencing pathways. Time-resolved small RNA sequencing opens new experimental avenues to deconvolute the timescales, molecular features, and regulation of small RNA silencing pathways in living cells.

Argonaute 结合的 microRNA 以动态和受调控的方式沉默 mRNA 表达，以控制有机体发育、生理和疾病。我们利用代谢小 RNA 测序来全面了解果蝇细胞内 microRNA 动力学。根据生物发生和衰变的绝对速率，microRNA 属于产生速度最快、寿命最长的细胞转录本之一，在稳态下每个细胞可处理多达 10 ⁵个拷贝。成熟的 microRNA 在几分钟内产生，揭示了生物发生的紧密细胞内耦合，该耦合被 pre-miRNA 尿苷化选择性破坏。对 Argonaute 蛋白稳态的控制会产生一个动力学瓶颈，该瓶颈与非编码 RNA 监视相配合，以确保忠实的 microRNA 加载。最后，受调节的小RNA衰减使得Ago1结合的microRNA能够选择性地快速周转，但不能使Ago2结合的小干扰RNA (siRNA)选择性地快速周转，这反映了小RNA沉默途径的鲁棒性的关键差异。时间分辨小RNA测序开辟了新的实验途径，以解开活细胞中小RNA沉默途径的时间尺度、分子特征和调控。