Insights into function and regulation of microRNAs by decoding degradation dynamics [serum]. Mus musculus

The regulation of the miRNA pool is one of the critical events in the definition of cell identity and behavior both in steady-state condition as well during cell transitions. While mechanisms controlling biogenesis have received much attention, little is known about how miRNA are turned over, due to the lack of proper approaches to analyze miRNA decay without globally perturbing gene transcription or miRNA biogenesis. To overcome such limitations we developed a “pulse-chase” approach for the analysis of miRNA degradation dynamics at genome-wide scale in unperturbed mammalian cells. MiRNA half-lives appeared heterogeneous: while many species were very stable (T1/2 >24h, ‘slow’ miRNAs), some others are quickly turned over (T1/2 =4-14h ‘fast’ miRNAs). ‘Fast’ species comprises passenger miRNAs, as expected, and a consistent number (25/129) of guide miRNAs. These miRNAs are associated to high Target:miRNA stoichiometric ratios, particularly evident considering targets with extensive complementarity, and are enriched for 3’-end modifications (tailing and trimming), implying that target abundance and enzymatic activities are in place and mediates miRNA degradation. By integrating decay rates with biosynthetic parameters, we could model the abundance and regulation of miRNAs as a function of transcription and degradation rates and highlight how different decays could affect the magnitude and the kinetics of miRNA regulation. These findings were recapitulated in a specific biological process, namely the regulation of miRNAs by serum stimulation of quiescent fibroblast, pointing out how multiple regulatory mechanisms (synthesis, decay, target abundance and miRNA modification) concomitantly control miRNA functions. Overall design: miRNA dynamic regulation in quiescent fibroblasts upon serum stimulation. Two independent biological experiments were performed over 9 time-points