Isolation and Quantification of mRNAs from Subcellular Phase-Separated Structures from Detergent Permeabilized Brain Cells

Abstract

Post-transcriptional regulation by RNA processing bodies, also known as P-bodies (PBs), is crucial for mRNA translation, localization, and stability in various animal cells, including neurons and glial cells. PBs facilitate spatial regulation of protein synthesis, influencing differentiation and synaptic function. Understanding mRNA dynamics in PBs is vital and is suspected to be impaired in neurodegenerative disorders. We developed a detergent-based method to isolate phase-separated P-bodies (PBs) free from cytosolic factors and RNAs, which allows us to examine their mRNA content under altered conditions. Using neuronal and glial cell models, we investigated the impact of A{beta}-oligomers on PB-associated mRNAs. In neurons, exposure to amyloid proteins disrupted the release of neuronal differentiation-related mRNAs, hindering their translation. Glial cells exhibited increased levels of cytoplasmic pro-inflammatory cytokine mRNAs after amyloid treatment, as they escaped from RNA processing bodies (PBs), ensuring enhanced translation. These findings underscore the dual role of PBs in controlling mRNA dynamics across cell types and illustrate how amyloid-induced stress may differently affect PB-mediated post-transcriptional control in neurons and astroglia. This method provides a platform for studying the mechanism and quantifying sub-organellar mRNA localization, as well as its effects on altered gene expression caused by amyloid proteins. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/669016v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@335e76org.highwire.dtl.DTLVardef@7ecf8dorg.highwire.dtl.DTLVardef@16c375dorg.highwire.dtl.DTLVardef@1a6c086_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA detergent-based differential solubilization of RNAs from PBs in brain cells. C_LIO_LIIn neuronal cells, {beta}-Amyloid oligomers target specific neuronal differentiation-related mRNAs to PBs. C_LIO_LIIn astroglia, {beta}-amyloid triggers the escape of inflammatory cytokine mRNAs from PBs. C_LIO_LIAltered mRNAs -protein phase separation connects PBs to neuroinflammation. C_LI