Fig. 5

Inhibition of IRE1 activity during ER stress decreases pre-miRNA stability in the cytosol. RNA half-life measurements were taken in 16HBE14o- exposed to Tm (2.5 µg/ml) in the presence or absence of 20 µM 4µ8C, and from the cells cultured in control conditions. Actinomycin D was added to stop transcription, after which the cells were collected, and total RNA was isolated from nuclear and cytosolic fractions. The levels of total hsa-miR-301a-3p (A), total hsa-miR-106-5p (B) nuclear pre-miR-301a (C), nuclear pre-miR-106b (D), cytosolic pre-miR-301a (E) and cytosolic pre-miR-106b (F) at each time point were measured by real-time PCR and normalized to endogenous 18 S rRNA levels. RNA values for each time point were calculated from 2 individual samples generated in at least 2 independent experiments and measured in 4 technical replicates. Relative RNA levels at the time points indicated were plotted as differences from RNA levels at the initial time point (t = 0). The mRNA half-lives were calculated from the exponential decay using the trend line equation C/C₀ = e^–kdt (where C and C₀ are RNA amounts at time t and at the t₀, respectively, and k_d is the RNA decay constant). The error bars represent SD. The calculated parameters are provided in Supplemental Table 1