Time-resolved small RNA sequencing unravels the molecular principles of microRNA homeostasis
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.
Stefan L. Ameres earned his Master’s degree in Biology at the Friedrich-Alexander University Erlangen-Nuremberg (Germany), and his PhD degree at the University of Vienna (Austria). In his PhD thesis, he reported the first in-depth enzymatic characterization of the human RNA interference effector complex. During his postdoctoral studies at UMass Medical School (USA), he discovered a novel pathway for the sequence-specific destruction of microRNAs in flies and mammals, establishing a potential therapeutic approach for microRNA-related diseases. In 2012 he joined IMBA (Vienna, Austria) as a group leader, where his lab uses innovative transcriptomics to study fundamental biological mechanisms of gene regulation.
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