Single-granule profiling reveals that RNP granule pH marks cellular translation

Area of Science:

• Cell Biology
• Biophysics
• Molecular Biology

Background:

• Ribonucleoprotein (RNP) granules are crucial for RNA metabolism in eukaryotes.
• Their biophysical properties, including pH, are poorly understood due to a lack of specific probes.
• Targeting these dynamic condensates is essential for understanding cellular processes.

Purpose of the Study:

• To develop a novel probe for mapping the pH of RNP granules, specifically processing bodies (P-bodies) and stress granules (SGs).
• To investigate the spatial pH heterogeneity within these granules at a single-granule level.
• To assess the probe's utility as a marker for cellular stress and translation activity, and for screening potential therapeutic agents.

Main Methods:

• Development of RNP granule-pH (RNPg-pH), a ratiometric and targetable fluorescent probe.
• Application of RNPg-pH to map pH within P-bodies and SGs in eukaryotic cells.
• Induction of cellular stress (oxidative, hyperosmotic, translational inhibition) to observe pH changes.
• Treatment with compounds like myrtenol and oridonin to assess their effects on stress-induced acidification and translation.

Main Results:

• RNPg-pH successfully mapped spatial pH heterogeneity within RNP granules, showing an alkaline periphery and acidic core near the nucleus.
• Cellular stresses induced granule acidification and correlated with reduced translation levels.
• RNPg-pH demonstrated sensitivity to translation activity.
• Myrtenol and oridonin reversed H2O2-induced acidification, restored translation, and delayed senescence.

Conclusions:

• The RNPg-pH probe provides a novel approach for monitoring RNP granule biophysics and function.
• Granule acidification is linked to cellular stress and impaired translation.
• RNPg-pH shows potential for screening anti-aging reagents and understanding RNP granule roles in RNA metabolism and cell function.