Degradation dynamics of microRNAs revealed by a novel pulse-chase approach

4sU pulse-chase RNA labeling and miRNA decay rates. (A) The scheme illustrates the experimental strategy of the 4sU pulse-chase approach used to follow miRNA decay dynamics. (B) The yield of 4sU-RNA at the indicated chasing times is reported. Forty micrograms of labeled total RNA was used at each time point. The dashed line marks the background level (yield of the negative control). (C) The half-life of Myc was measured by RT-qPCR using the pulse-chase protocol according to a one-phase exponential fitting (N = 2). Data (log₂ scale) were normalized on the levels of Actb, a highly stable gene (Yang et al. 2003). (D,E) Half-lives of mRNAs, pri-miRNAs, small RNAs (D), and mature miRNAs (E) were estimated as in C. Shown is the average and the SEM of two independent experiments. The miRNA data were normalized to the levels of Snord72, a highly stable short RNA. (F) Half-lives calculated using the pulse-chase approach (shown in D,E) were compared with those indirectly determined by 4sU pulse labeling, as described previously (Dolken et al. 2008). A linear correlation was calculated for mRNAs/pri-miRNAs (red dashed line) or miRNAs (yellow dashed line). Shown also is the coefficient of determination (R²) of the linear fitting. (G) Canonical miRNA species analyzed in the “decay data sets,” distinguished as guide or passenger miRNAs according to miRBase and TargetScan databases. (H) Distribution of miRNA half-lives. The red and blue lines mark boundaries of fast- and slow-decaying species. (I) Half-lives (mean with confidence interval) are plotted against miRNA expression (copies per cell [CPCs]; log₁₀ scale). (J) Degradation dynamics of fast (N = 25, T_1/2 < 14 h) and slow (N = 32, T_1/2 > 24 h) guide miRNAs are shown along the time courses. Data were normalized to miR-92a-3p and reported as log₂ fold-change over the 0-h time point. (K) The frequency of passenger versus guide miRNAs within the decay classes has been analyzed by contingency (χ² = 6.24, P = 0.042). Ambiguous species were not included. (L) Box plot shows the distribution of half-lives for matched guide and passenger species. P-value (Wilcoxon test) is shown.