Time-resolved small RNA sequencing unravels molecular principles of microRNA homeostasis [Ago1OE + TailorKO]

Argonaute (Ago)-bound microRNAs (miRNAs) silence mRNA expression in a dynamic and regulated manner to control organismal development, physiology and disease. Here, we employed metabolic sequencing of small RNAs for a comprehensive view on intracellular miRNA kinetics in Drosophila. Based on absolute biogenesis and decay rates, miRNAs rank among the fastest produced and most long-lived cellular transcripts, enabling them to reach >105 copies per cell at steady-state. Tight coupling of steps in biogenesis produces mature miRNAs within minutes and is effectively disrupted by pre-miRNA uridylation. In contrast, control over Ago protein homeostasis generates a kinetic bottleneck that cooperates with ncRNA surveillance to ensure faithful miRNA loading. Finally, regulated small RNA decay enables the rapid turnover of specific Ago1-bound miRNAs but not of Ago2-bound siRNAs, reflecting key differences in the robustness of small RNA silencing pathways. Our work opens new experimental avenues to deconvolute the timescales, molecular features, and regulation of small RNA silencing pathways in living cells. Wild-type Drosophila S2 cells, TailorKO, FM-tagged Ago1 overexpression in wild-type background, Ago2KO, and FM-tagged Ago1-overexpression in Ago2KO background were subjected to biochemical purification of Argonaute ribonucleoprotein complexes and smallRNA sequencing in 2 or 3 replicates (as indicated). The libraries were first aligned to S2 viral genomes to eliminate contaminants, then to dm6 to determine mappers in ncRNA and coding regions in a hierarchical manner (rRNA, mitochondria, tRNA, snRNA, snoRNA, endo-siRNA, cisNAT, miRNA, intronic, exonic, TE, none).