Probing the Role of RNA Helicases in Modulating Stress Granules, RNA Localization, and P-Body Docking.

Stress granules (SGs) are cytoplasmic, membrane-less ribonucleoprotein (RNP) granules that form in response to cellular stress following the inhibition of translation initiation. Their assembly, composition, and crosstalk with processing bodies (P-bodies) are mediated by protein-protein, RNA-protein, and RNA-RNA interactions. DEAD/DEAH-box RNA helicases are key regulators of stress granules. Here, we present standardized protocols for (i) immunofluorescence of stress granule and P-body scaffold proteins and helicases, (ii) single-molecule RNA fluorescence in situ hybridization (smRNA FISH), and (iii) quantitative image analysis using Imaris software, including single-cell and nuclear segmentation, stress granule and P-body surface rendering, docking interaction analysis, and RNA spot detection.