Screening of small molecules affecting mammalian P-body assembly uncovers links with diverse intracellular processes and organelle physiology

Abstract

Processing bodies (P-bodies) are cytoplasmatic mRNP granules containing non-translating mRNAs and proteins from the mRNA decay and silencing machineries. The mechanism of P-body assembly has been typically addressed by depleting P-body components. Here we apply a complementary approach and establish an automated cell-based assay platform to screen for molecules affecting P-body assembly. From a unique library of compounds derived from myxobacteria, 30 specifically inhibited P-body assembly. Gephyronic acid A (GA), a eukaryotic protein synthesis inhibitor, showed the strongest effect. GA also inhibited, under stress conditions, phosphorylation of eIF2α and stress granule formation. Other hits uncovered interesting novel links between P-body assembly, lipid metabolism, and internal organelle physiology. The obtained results provide a chemical toolbox to manipulate P-body assembly and function.

Keywords: P-body assembly; eIF2α; gephyronic acid A; inhibitors; myxobacterial metabolites; processing bodies; stress granules.

Figure 1. Distribution of (A) chemical classes and (B) known activities for all 30 compounds found to effectively reduce P-body formation with no more than 30% reduction in cell viability.

Figure 2. The translation inhibitor gephyronic acid A strongly reduces P-body formation in HeLa cells. (A) Cells were treated with either 1 µM of gephyronic acid A (GA) for 3.5 h, 25 µg/ml of cycloheximide for 15 min, 100 µg/ml of puromycin for 1 h, or mock-treated. After the indicated time, cells were immunostained with an antibody for DDX6 (green) and the nuclei was visualized with DAPI (blue). The number of DDX6-containing P-bodies from at least 100 cells per condition was quantified and related to those detected in mock-treated cells. Representative images for each condition are shown. Numbers below the panel show the median and the standard error of the mean of the P-body number per cell relative to the untreated control (set to 100%). (B) HeLa cells were incubated for 1 h (upper graph) or 3.5 h (lower graph) with 0, 50, 100, 500, or 1000 nM of GA. Cells were immunostained to quantify DDX6- and Dcp1-containing P-bodies in at least 100 cells. The relative abundance of DDX6- and Dcp1-containing P-bodies vs. log GA concentration is shown related to mock-treated cells. Right graphics indicate cell viability for the different concentrations of GA and times of incubation using an ATP assay that measures intracellular ATP levels. Error bars indicate standard error of the mean. (C) For both time-points, expression levels of DDX6 and Dcp1 were tested by western blot under the higher concentrations of GA. β-actin was used as a control for protein loading.

Figure 3. GA prevents stress granules formation and inhibits P-body assembly when stress conditions are applied. (A) HeLa cells were either mock-treated, treated with 500 nM of gephyronic acid A (GA) for 3.5 h, treated with 0.1 mM of arsenite (Ar) for 30 min, or treated with GA for 3.5 h adding plus Ar during the last 30 min. After the indicated times, cells were immunostained with an antibody for the stress granule component eIF3 (upper panel) or the P-body component DDX6 (lower panel). (B) Protein levels of eIF3, DDX6, eIF2α, and eIF2α-phosphorylated were tested by western blot using β-actin as a control for protein loading. Numbers below the panel indicate the percentage of eIF2α-phosphorylated expression levels relative to those detected in arsenite-treated cells.

Figure 4. In stressed cells, GA promotes disassembly of SGs but not of P-bodies. HeLa cells were either mock-treated, treated with 0.1 mM of arsenite (Ar) for 1.5 h, or treated with Ar for 1.5 h adding GA 500 nM during the last hour. After the indicated times, cells were immunostained with an antibody against the stress granule component eIF3 (upper panel) or the P-body marker DDX6 (lower panel).