Analysis of interferon-beta mRNA stability control after poly(I:C) stimulation using RNA metabolic labeling by ethynyluridine

Abstract

Interferon-beta (IFN-β) is a critical antiviral cytokine and is essential for innate and acquired immune responses to pathogens. Treatment with polyinosinic:polycytidylic acid (poly(I:C)) induces transient accumulation of IFN-β mRNA, which involves an increase and a decrease of IFN-β mRNA. This phenomenon has been extensively analyzed as a model for understanding the mechanisms of gene induction in response to external stimuli. Using a new RNA metabolic labeling method with ethynyluridine to directly measure de novo RNA synthesis and RNA stability, we reassessed both de novo synthesis and degradation of IFN-β mRNA. We found that transcriptional activity is maintained after the maximum accumulation of IFN-β mRNA following poly(I:C) treatment on immortalized human bronchial epithelial cells. We also observed an unexpected change in the stability of IFN-β mRNA before and after the maximum accumulation. The results indicate that this method of RNA metabolic labeling provides a general approach for the simultaneous analysis of transcriptional activity and mRNA stability coupled with transcriptional timing.

Activation of Type I and III Interferon Response by Mitochondrial and Peroxisomal MAVS and Inhibition by Hepatitis C Virus

“…Consistent with this result we observed induction of endogenous ISG56, IFN-β and type III IFN expression upon SeV infection and these responses barely differed between the MAVS variants ( Fig 3C ). As reported earlier [ 43 , 44 ], IFN-β expression was rapidly and very transiently induced and this also applied to IFN-λ1. In contrast, activation of IFN-λ2/3 expression was much more sustained and did not decline throughout the 48 h observation period.…”

The RNA Degradation Pathway Regulates the Function of GAS5 a Non-Coding RNA in Mammalian Cells

“…We suspect that BrU is not effectively incorporated into serum-starved cells. Instead, we used another inhibitor-free method, 5-Ethynyluridine (EU) pulse-labeling, which is commercially available as the Click-iT Nascent RNA Capture Kit (Invitrogen) [33]–[36]. We have successfully determined the half-life of GAS5 with or without serum starvation by the EU method, and found that t 1/2 of GAS5 was prolonged from 4.2 h to >12 h in serum starved cells (Figure 4B).…”

“…Analysis of RNA half-lives was performed by EU pulse-labeling of RNA using the Click-iT Nascent RNA Capture Kit (Invitrogen) [33]–[36]. After serum withdrawal for 24 h, 200 µM of EU was added to medium and cells incubated for an additional 24 h. At indicated time points after replacing EU-containing medium with EU-free medium, cells were harvested.…”

“…However, inhibitor-mediated global transcriptional arrest has a profound impact on cellular physiology, and affects RNA decay rates [7], [23], [32]. Thus, we used a novel inhibitor-free method of pulse-labeling nascent transcripts with 5′-bromouridine [7], [26], [33] or with 5′-ethynyluridine [33]–[36] to enable the measurement of RNA decay under non-disruptive conditions. We found that GAS5 levels were elevated owing to prolonged decay rates in response to UPF1 depletion, and that multiple apoptosis-related genes were downregulated.…”

Metabolic labeling and recovery of nascent RNA to accurately quantify mRNA stability

“…We note that other nucleoside analogs can be used for metabolic labeling and half-life determination including 5-bromouridine (5BrU) [33–36] and 5-ethynyluridine (5EU) [37]. The approach described here could be adapted to these nucleosides simply by replacing the positive control RNA with one labeled with 5EU or 5BrU and following established protocols for labeling and fractionation.…”