Time-resolved small RNA sequencing unravels molecular principles of microRNA homeostasis [ago2KO s4U TT-SLAMseq]

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. Overall design: Combination of TT-seq with SLAM-seq, labelling Ago2KO Drosophila S2 cells for 5 minutes. Labelling was performed in three biological replicates, each replicate was once sequenced only as "input" and once as "bound" (TT-seq purified). Both "input" and "bound" underwent SLAMseq IAA treatment. Unlabelled input was sequenced once (prepared with NEBNext Ultra II Directional RNA Library Prep Kit). All Samples were analysed with slamdunk v0.3.4